RU2238324C2 - Universal vector for stable transformation of chloroplast genome, method for stable transformation of plant-target, method for conferring to plant resistance to plant-target against herbicide, method for assay of transformation of chloroplast and stably transformed chloroplast genome - Google Patents

Abstract

FIELD: genetic engineering of plants, agriculture.

SUBSTANCE: vector involves the expression cassette comprising a sequence encoding the interesting peptide that is joined with regulatory sequences providing expressing of the encoding sequence in chloroplasts of plant-target. Cassette-flanking sequences are homologous with conservative spacer sequences of chloroplast genome of target that provides the stable integration of encoding sequence into chloroplast genome. Insertion of mentioned vector into genome of plants of different species allows conferring to them new properties that are stably inherited in progeny.

EFFECT: valuable biological properties of vector, improved transformation and assay.

29 cl, 62 dwg, 2 tbl, 16 ex

Description

This application claims the benefit of the pending application under registration number 60/055314, registered August 7, 1991, incorporated by reference in this application. This application also claims the benefit of the pending provisional application under registration number 60/079042, registered March 23, 1998, entitled "Universal chloroplast integration and expression vector, transformed plants and products thereof," incorporated by reference in this description. In addition, this application is a partial continuation of the pending patent application under registration number 08/591407, registered January 25, 1996, Henry Daniell, which is a partial continuation of the application under registration number 08/591407, registered March 18, 1994, now abandoned, which, in turn, it is a continuation of the application under registration number 07/249616, registered on September 26, 1988, now abandoned.

FIELD OF THE INVENTION

This application relates to the field of genetic engineering of plant genomes, in particular to genetic engineering of the plastid genome, such as chloroplasts, and to the sustainable transformation of the chloroplast genome of a plant of any kind.

Related technical solutions

This application relates, in particular, to a universal chloroplast expression and integration vector competent for transforming any plant with one or more genes of interest. A previously registered patent application under registration number 08/591407 describes plant cells transformed with an expression cassette containing an exogenous DNA sequence that is stably integrated (covalently linked) with the chloroplast genome of the target plant cell. "Stably" integrated DNA sequences are sequences that are inherited through replication of the genome by daughter cells or organisms. This resistance is manifested through the ability to create constant cell lines, clones or transgenic plants that make up a population containing exogenous DNA.

Similarly, US Pat. No. 5,693,507 (1997), Danielle and McFadden, describes robust integration using an expression cassette containing an exogenous DNA sequence encoding a desired trait and transformed plants.

BACKGROUND OF THE INVENTION

Advantages of chloroplast transformation over nuclear transformation.

The attractiveness of the transformation of the chloroplast genome before the transformation of the nuclear genome is associated with serious dangers arising from the latter. A well-known concern is the leakage of foreign genes during the dispersal of pollen from transgenic cultivated plants to their wild related forms. It has been shown that transgenic pollen will deliver foreign (transgenic) genes to other sexually compatible plants (which is determined by the prevalence of the marker gene in offspring collected from non-transgenic plants grown in adjacent areas). For example, pollen was dispersed from a central experimental plot containing transgenic cotton plants to surrounding non-transgenic plants at different distances in different directions (Lewellyn and Fitt, 1996; Umbeck, P.F., et al., 1991). In addition, the concentration of marker genes in wild sunflower averaged about 28-38%; in wild strawberries, growing within 50 meters of the site with strawberries, over 50% of wild plants contained marker genes from cultivated strawberries (King, J., 1996) .;

The leakage of foreign genes through pollen is especially worrying for the environment in the case of herbicide resistance genes due to the high degree of flow from cultivated plants to wild related forms. It is alarming that leakage of genes from transgenic cultivated plants to their wild related forms will create super-weeds. For rice (Oryza sativa), a flow of genes from cultivars to wild related forms was noted - to O. perennis (Barrett, 1983) and to red rice (O. sativa; Langevin et al., 1990). In the south of the United States, red rice becomes the main weed because the herbicides that infect it also infect cultivated rice. Red rice is contaminated with lower prices. Some researchers introduce the bar gene into cultural rice, which confers resistance to glufosinate (Liberty) in order to combat its wild form (Oard et al., 1996; Sankula et al., 1996). However, due to gender compatibility, the introduction of a gene expressed in the nucleus will allow transmission of a trait of such resistance to red rice through pollen.

Similarly, transgenic oilseed rape, which was genetically engineered to be resistant to herbicides, was inadvertently crossed with the wild related form of Brassica campestris (field mustard) and rendered herbicide resistant even in the first generation from backcrossing in the field (Mikkelson, TR, et al. , 1996).

The maternal heredity of the introduced genes prevents the leakage of genes through pollen. The introduction of foreign genes through chloroplast genomes (which are maternally inherited in the case of most cultures) is a solution to this problem. Also, enzymes or target proteins for most herbicides (for example, amino acid and fatty acid biosynthesis pathways or photosynthesis) are compartmentalized within the chloroplast. Another important advantage of chloroplast transformation is a higher level of expression of foreign genes due to the very high number of copies (5000-10000) of chloroplast genomes in plant cells. Since the mechanism of transcription and translation of the chloroplast is prokaryotic in nature, resistance genes for herbicides of bacterial origin can be expressed in chloroplasts at an extremely high level.

Transformation of the chloroplast genome.

Early studies on chloroplast transformation focused on the development of organelle systems using intact chloroplasts capable of efficient and long-lasting transcription and translation (Daniell and Rebeiz, 1982; Daniell et al., 1983) and the expression of foreign genes in isolated chloroplasts (Daniell and McFadden , 1987). These experiments were carried out on the basis of the premise that it is possible to introduce isolated intact chloroplasts into protoplasts and regenerate transgenic plants (Carlson, 1973). The discovery of the gene gun as a tool for transformation provided an opportunity for direct plastid transformation in plants (Daniell, 1993). Using the gene gun, unstable expression of foreign genes in dicotyledonous plastids (Daniell et al., 1990; Ye et al., 1990), monocotyledonous ones (Daniell et al., 1991), long-term expression of foreign genes using independently replicating chloroplast expression vectors (Daniell et al., 1990) and the sustainable integration of a selectable marker into the chloroplast genome of tobacco (Svab and Maliga, 1993). Tobacco plants resistant to certain insects are obtained by integrating the cryIAc gene into the chloroplast genome of tobacco (McBride et al., 1995; US Pat. No. 5,451,513, incorporated herein by reference). Sustainable transformation of plastids of higher plants is carried out so far only in tobacco.

Famous studies of the chloroplast genome.

As for the sustainable integration of a foreign gene into the chloroplast genome of a higher plant, today there are reports of such integration only in tobacco. This is achieved using a tobacco specific vector derived from the chloroplast genome of tobacco; those. the vector contained a sequence homologous only to the chloroplast genome of tobacco, which is not highly conserved in the chloroplast genomes of other plants. Such a vector is not suitable for the stable transformation of plants other than tobacco species. The only published report on the expression of a foreign gene in a form of a plant other than tobacco belongs to the leaves and seeds of wheat (Daniell et al., 1991), but stable integration has not been carried out. Sustainable integration of a foreign gene into the chloroplast genome of a monocotyledonous plant has never been reported. At least, the previously described transformation / regeneration schemes in cereals (monocotyledonous) cannot be adapted to the transformation of plastids due to the inefficiency of inheritance within such systems. Sequential / serial selections (re-selections) (Daniell, 1997), which were also considered important for achieving homoplasmy, cannot be performed using such used regeneration systems. Recently developed unique transformation / regeneration schemes for maize (Rudraswamy, 1997) and rice (not published) have the potential for manifesting significantly increased efficiency and allow for more than one selection cycle during regeneration.

US Pat. No. 5,451,513, Maliga et al., And Svab et al., 1990, describe the transformation of the tobacco plastid genome using a non-lethal selection method that uses plastid DNA encoding a non-lethal breeding phenotype. According to Maliga et al., It is precisely non-lethal selection that is necessary to obtain transplagenic lines.

In contrast to the method of Maliga et al. The method of the invention relates to selection, which is lethal to all non-transformed plants, with the exception of tobacco. Only transformed plants survive and continue to grow. Such lethal selection occurs with almost all antibiotics, including spectinomycin and streptomycin, in a medium containing an antibiotic at a concentration of 500-1000 μg / ml. Maliga et al. showed that such conditions are not fatal to tobacco. Moreover, unlike the method of Maliga et al. in accordance with the method of the invention, transformations to homoplasmy can be achieved even in the first selection cycle.

European Patent Application No. 0251654, Cannon et al., Describes a transposon-mediated transformation of tobacco chloroplasts, for example, using the bacterial transposon Tn5. A vector containing a transposon is sent to the chromosomal region, known as the "transcriptionally silent" region, in order to preserve the transcriptional integrity of the native genes. Such a transcriptionally silent region is identified as the region localized between two divergent promoters of chloroplast genes, for example, promoters for genes for the large chloroplast subunit of ribulose bisphosphate carboxylate (RbcL, ″ LS RuBisCo ″) and for β-ATPase (atpB). These promoters transcribe genes in opposite directions from the silent region of the chromosome. Cannon et al. they do not describe the transcription terminator in the expression vector, but it is known that such terminator regions are necessary for gene expression in plastids. Finally, it is not shown that Cannon et al. carried out a stable transformation of chloroplasts.

The invention described here has several signs of difference from the method of Cannon et al. The invention describes a stable transformation transmitted to offspring. Integration is not directed to the transcriptionally inactive region of the chloroplast chromosome. According to the invention, the cassette (containing the transcription terminator, as described hereinafter) is integrated into the transcriptionally active region of the chloroplast genome. Promoters regulate the expression of one or more genes. In contrast to the method of Cannon et al. in accordance with this invention, the transposon is not involved in the transformation of the chloroplast.

A publication in NATO Asi Series, Daniell et al., 1994 reports resistance to harmful insects through chloroplast genomes, and demonstrates the expression of the CryIIA protein in insect control plants. Communication by Daniell et al. support the work of McBride et al., 1995, and US patent 545818 (1996), which describes the expression of the CRYAc protein of Bacillus thurin-giensis in plastids. The vectors described by McBride are designed to introduce the construct only into the chloroplast genome of tobacco.

The need for a vector for the transformation of various plants.

From the prior art it is obvious that there is an urgent need for a chloroplast integration and expression vector for the transformation, preferably stable, chloroplast genomes of many different plant species. Such a “universal vector” would allow the transformation of the chloroplast genome of the selected target plant with a heterologous (foreign) coding DNA sequence and eliminate the need to create vectors, each of which is specifically suitable for the transformation of the chloroplast genome of a particular plant species that needs to be transformed.

The problem of creating such a universal vector that meets the requirements of the transformation of various plants is familiar to the inventors, but has not yet been solved.

Representations of the prior art about the intergenic spacer region.

While the nucleotide sequence of the coding regions of the genome, including the chloroplast genome, is often conservative among species, sequences flanking functional genes, i.e., the spacer regions between coding regions, on the contrary, are generally not conservative. The recognized opinion about the loss of conservatism and, thus, a low degree of homology between species of spacer regions is that spacer regions, as a rule, do not perform essential functions. Consequently, there is a small, if not absent, selection pressure to maintain the sequence of spacer regions among species. The sequence of spacer regions can change without undesirable effects.

Stummann et al., 1988, report that the gene sequence of the operon of the ribosomal RNA of the chloroplast genome is the same in different plant species, including tobacco, maize and the liver (Marchantia), and that the coding sequences of this operon are highly homologous. Stummann also reports that the interspecies homology of the operon is less than the interspecies homology of the coding regions of genes. This fact is consistent with the loss of conservatism in the spacer regions and means that the interspecies homology of the spacer regions in the ribosomal RNA operon is relatively low.

In contrast to the conservativeness of the spacer regions, the invention uses spacer regions that are highly conserved among different plants to create vectors competent for transforming various plants.

General idea of the invention

The invention relates to universal chloroplast integration and expression vectors that are competent for the stable transformation and integration of genes of interest into the chloroplast genome of many plant species. The invention relates to transformed plants and their offspring. Monocotyledonous and dicotyledonous plants that have never been transformed before are transformed. Plants are transformed with a synthetic gene for the expression of valuable biodegradable polymers based on proteins (PBP). Transformed plants produce very valuable molecules. Crops are given resistance to the main classes of chemical herbicides. Herbicide resistance is used as a lethal breeding marker for the transformation of chloroplasts. Transformed plants are able to express, in addition to the intended trait, the desired secondary unintended trait. Transformed plants are given resistance to insects, both in relation to insects sensitive to Bt-toxins, and in relation to insects that have developed resistance to Bt-toxins.

Summary of the invention

Interspecific spacer region. Concept of the invention.

It was found in contrast to the generally accepted opinion that the chloroplast (ct) genome of plants contains spacer regions with very conservative nucleotide sequences. The highly conserved nature of the nucleotide sequences of these spacer regions of the chloroplast genome makes these spacer regions found to be ideal for creating vectors for the transformation of chloroplasts of various plant species and eliminates the need to create separate vectors for different plants or individual cultivated species, which might require first determining the DNA sequence each of the chloroplast genomes. This discovery has many beneficial consequences and is important for practical applications.

Some embodiments of the invention

Universal vector.

The invention has several useful embodiments. The invention relates to a universal integration and expression vector, hereinafter referred to as "UV", and its use for the expression of at least one phenotype in many different plants.

The integration expression universal vector of the invention contains an expression cassette (also described below) containing the necessary genetic elements for unstable or preferably stable transformation of plastids, for example, the chloroplast genome, of a target plant cell from a foreign (heterologous) DNA encoding a molecule of interest, as a phenotype that expressed by a plant or non-plant highly valuable molecule similar to a biologically active peptide (or polypeptide). A universal vector is created with the transcriptionally active region of the chloroplast genome, which is highly conserved in many chloroplast genomes of higher plants. Preferably, this region is a spacer 2, an intergenic spacer region between the t-RNA ^Ile and the tRNA ^Ala region. Such a region here refers specifically to the “spacer” region, since in the chloroplast genome it is intergenic between several genes in the rRNA operon, which is transcribed by one promoter. When such a region is inserted into a universal vector, it is commonly referred to herein as a “borderline” or, preferably, “flanking sequence” or “flanking sequences”. This is so because in the universal vector operably linked genetic elements for stable transformation of the target plant plastids are flanked on each side by a sequence, i.e. fragment of the spacer region. Flaming sequences in the vector and spacer sequences in the chloroplast genome have sufficient homology with each other to undergo homologous recombination. A universal vector is inserted into the spacer of the transcriptionally active region in the chloroplast genome. Typically, the spacer region is located in the region of the inverted repeat of the chloroplast genome. The rest of the structure, i.e. in addition to flanking sequences and expression cassettes, they are generally referred to herein as a “vector” containing bacterial sequences similar to the plasmid cloning vectors pUC, pBR322, pGEM or pBlueScript.

Expression vector or cassette.

A universal vector contains an expression cassette that is flanked on each side by a flanking sequence. A suitable expression cassette for use in the invention is described in US Pat. No. 5,693,507 (1997), incorporated herein by reference. Such a cassette contains operably linked transcription initiation region, functional in the plant chloroplast, at least one heterologous DNA sequence encoding a target molecule of interest, for example, a gene (or its functional part) encoding a biologically active compound, and regular sequences located in front of 5′- and after 3′-ends, and the transcription termination region, which provide expression of the coding sequence in the chloroplast genome of the target plant. Preferably, the expression cassette is flanked by plant DNA sequences similar to the chloroplast DNA sequences in order to facilitate the stable integration of the expression vector into the chloroplast genome. In the design of the expression cassette, the DNA sequence contains one or more cloning sites for the integration of the gene (s) of interest.

Spacer sequences identified in plastids of higher plants are universally conserved among a wide variety of plants. It was found that these sequences are ideal for creating universal vectors of the invention, which, as a result, are competent for transforming the chloroplast genome of the most diverse (or many) target plants through homologous recombination. Thus, it does not matter from which individual spacer of a particular plant a universal vector is created.

As you know, as a rule, it will be advisable to have at least one additional heterologous nucleotide sequence encoding a selected phenotype, such as a gene that provides resistance to antibiotics, or its functional part, which will serve as a marker associated with the expression cassette or universal integration expression vector. This facilitates the identification of plant cells into which a foreign gene is stably integrated. Marker genes are known from the literature, for example β-lactanase, herbicide resistance genes such as the psbA mutant gene, or EPSPS-aroA, the cat gene encoding chloramphenicolacetotransferase, and the uidA gene encoding β-glucuronidase (gus), and others.

It turned out that tobacco is unique in its immunity to the lethal effects of streptomycin and spectinomycin. Although tobacco leaves lose pigmentation when exposed to an environment with such antibiotics, continued growth can be observed. However, this property of tobacco is easily deceived. There are many available lethal antibiotics for tobacco, such as hydromycin. In another approach, a gene that expresses a visible marker similar to color, fluorescence and the like similar to the mGFP reporter gene encoding a green fluorescence protein should be selected.

The method of transformation.

The invention relates to a transformation method that can provide homoplasmy (integration of foreign genes into all chloroplast genomes of a plant cell) after the first selection cycle without the need for a further selection process. The plant transformation method uses a universal vector constructed with flanking sequences from a different kind of plant than the kind of transformable target plant. On the other hand, a vector can; contain flanking sequences from the same species as the target plant, including tobacco.

A way to create a universal vector.

The invention also relates to a method for creating a universal chloroplast integration and expression vector. In order to do this, it was found that the spacer part of the chloroplast genome from any plant is highly homologous in several plant species. The nucleotide sequence corresponding to this spacer region is obtained from the identified chloroplast genome (or synthesized) and introduced into a suitable vector, for example into a plasmid, by subcloning. The spacer region is located as flanking sequences to the expression cassette containing the necessary genetic elements for transformation of the plastid and expression of the foreign gene (s).

Any chloroplast transformation method can be used. Any gene (or its functional part) that can be used to transform the chloroplast and which encodes the desired peptide that gives the desired trait to the target plant is suitable for transformation using a universal vector.

Transformed plants. The invention also relates to plants in which the chloroplast genome is stable, i.e. permanently transformed by the universal vector of the invention, including their descendants.

The invention includes monocotyledonous plants similar to cereals, such as maize, rice, barley, oats, wheat and cereal, or plant cells, and their progeny in which the chloroplast genome is stably transformed by a universal vector derived from the same species as the transformed plant , or from another species. The invention relates to dicotyledonous and monocotyledonous plants, stably transformed in one selection cycle, due to homoplasmy achieved using a universal vector containing a chloroplast replication origin (ori). The invention also relates to stably transformed plants of various species, including varieties of the same species, genera, families, orders and plant types.

According to the invention, a plant in which the chloroplast genome is stably transformed by one or more foreign genes of interest includes mature plants and their progeny, similar to seedlings and embryos. The term "plant" in this context also includes parts of the plant, such as explants like cuttings, tissue cultures, cell suspensions and calluses.

Thus, the invention includes stably transformed multicellular plants, their progeny, seeds and transformed plastids, for example, chloroplast and the like, and a method for regenerating transformed plants.

In this description and in the claims, when various ″ species ″ are mentioned, the term ″ species ″ refers not only to ″ species ″, but also to varieties within a species, genera, families, order and types of the plant world. Thus, it should be borne in mind that a universal vector that can be used to transform plants of various species is capable of transforming plants of various varieties within a species, various genera, various families, various orders, and various types. The term ″ plant ″ (or ″ plants ″) as used herein is intended to be generic.

Expression of non-plant products

Genes of biopolymers.

Another embodiment of the invention using a universal integration and expression vector relates to plants transformed with a synthetic biopolymer gene that encodes microbial degradable protein-based polymers (PBPs).

These polymers have important, practical properties described below.

Getting high-value molecules - biologically active molecules.

The discovery of interest that transformation with a synthetic gene is feasible to obtain HRV, which does not require a natural analogue in an animal or plant, demonstrates the wide applicability of the vector in yet another area of human activity - the production of biologically active molecules like pharmaceuticals in plants from any synthetic or natural gene or its functional part.

Therefore, another embodiment of the invention is the use of transformed plants as bioreactors (factories) for pharmaceuticals. There are at least two potential possibilities, usually necessary for the production of proteins or a valuable pharmaceutical product, which are not possible in prokaryotic systems. Plants, unlike bacteria, are capable of producing a foreign protein in a biologically active form. Plants are often also more tolerant of changes in their biosynthesis pathways. Thus, plants can be transformed with a gene that is non-functional in plants (or foreign to them), which can be synthetic or not, which can be normally functioning (competent) in animals (mammals), oviparous, in insects or other species.

The invention also relates to transformed plants containing a gene obtained using an expression cassette, preferably a universal vector that encodes many desired products, in particular biologically active molecules like peptides (polypeptides), proteins, insulin, human serum albumin (HSA), and other molecules, also described hereinafter. The plants are given the opportunity to grow, or cause their growth, and products are isolated from a transformed culture like tobacco, maize, etc., and, if necessary, first harvested and, if necessary, cleaned.

Herbicide tolerance.

Another important embodiment of the invention relates to transgenic herbicide resistant plants in which, preferably a stable, foreign transgene is introduced into the chloroplast genome using a universal vector, conferring resistance to one or more herbicides. Of particular importance are transformed plants that show glyphosate resistance and are thus resistant to ″ ROUNDUP ™ ″, a herbicide commercially available from Monsanto Company. The universal vector provides an effective means of transforming the chloroplast genome of any plant and imparting resistance (or tolerance) to any of the herbicidal chemicals.

Another aspect of the invention is a method for transforming a plant using an expression cassette, preferably using a universal vector, to cause it to produce an unintended (secondary or other) trait (or phenotype). (See, for example, Penazloza, V., et al. (1995), where it is reported that expression of the thromycin-β-phosphotransferase gene confers herbicide glyphosate resistance.]

In another aspect of the invention, herbicide resistance is used as a marker gene for the transformation of chloroplasts.

Resistant to insects. Another embodiment of the invention relates to insect resistance. With increasing concern about the use of chemical pesticides, the use of compositions with Bacillus thuringiensis (Bt) is widely promoted. Bacillus thuringiensis produces many types of crystalline inclusions that are toxic to insects. Proteins containing such inclusions are classified based on the range of host insects and protein homology. Toxins CRYI and CRYII have insecticidal activity against Lepidoptera or Lepidoptera and Diptera, respectively. CRYI protoxins have a size of 130-135 kDa and are enzymatically cleaved into 65 kDa proteins with an insecticidal effect. CRYII protoxin has a size of 65 kD with a protein with a molecular weight of 60-62 kD for insecticidal activity. Many pests of importance to the farm (especially the fire family) are susceptible to the CRYIIA toxin, including the corn moth Ostrinia nubilalis, the corn grinder Elasmopalpus lignosellus, the bean mump Maruca testulalis, the scoop Heliothis virescens sextra lentis mollus, Daniell et al., 1994.

However, compositions with Bt are not as effective as originally expected, due to their sensitivity to UV radiation, inadequate coverage, high cost and a limited range of hosts. Therefore, the delivery of Bt toxins via Bt transgenic plants is attractive.

An acceptable level of insect control occurs in the case of nuclear transgenic Bt cotton versus Heliothis virescens scoop, but these plants do not express enough Bt toxin to control the cotton scoop Helicoverpa zea. In addition, these Bt genes may be inadvertently transferred to related plants through pollen. In order to circumvent these difficulties, chloroplast transformation and expression were evaluated in accordance with the invention for the following reasons. 1) Plant cells containing chloroplasts can contain up to 10,000 copies of genes per cell, 2) chloroplasts can read intact bacterial DNA, including operons, and 3) chloroplast genomes are inherited maternally and, therefore, leakage of a foreign gene through pollen is strongly excluded, since DNA chloroplasts are usually destroyed in pollen.

CRY2A was chosen because CRY2A is relatively non-homologous to CRY1A and therefore exhibits only weak cross-resistance against some H. virescens resistant CRY1A populations. Since CRY2A is ″ shortened in nature ″, high levels of expression can be achieved without sacrificing the 3′-region.

Corresponding resistance for the chloroplast tobacco genome transformed by the universal vector of the invention has been achieved to insects that are inherently sensitive to Bt toxins, as well as to insects that have developed resistance or lower susceptibility to Bt toxins. Insects that never died under the influence of any CRY toxin showed 100% mortality on transformed tobacco.

Therefore, expression of cry2A in a plant can be an extremely useful tool in controlling many pests, and resistance to Bt is overcome at the same time. In chloroplasts, other insecticidal proteins, such as cholesterol oxidase, are expressed at a high level, which kill cotton weevil by a different mechanism.

In the future, other embodiments of the invention will become apparent.

Description of drawings

Figure 1 shows a map of the chloroplast genome of tobacco. Bold lines on the genome map represent regions of the inverted repeat of the chloroplast genome. The arrows labeled ″ UV ″ indicate insertion sequences for the preferred embodiment of the universal integration and expression vector (UV); the arrow labeled ″ TV ″ shows the insertion sequence for the tobacco vector (TV).

Figure 2A shows tobacco chloroplast vector (TV) pZS-RD-EPSPS for expression of herbicide resistance.

Figure 2B shows the universal chloroplast expression and integration vector (UV) pSBL-RD-EPSPS for expression of herbicide resistance.

Figure 3A shows the universal chloroplast integration and expression vector pSBL-CG-EG121 for expression of biopolymers.

Figure 3B shows integration and expression in tobacco and the pZS-CG-EG121 vector for expression of biopolymers.

Figures 4A-4D show the sequence homology of the spacer regions of tobacco and other crop species. The site for embedding a foreign gene is shown by arrows. In front of the foreign gene embedding site, the site of the origin of replication (ori) is shown.

Figures 4E-4G show the aligned 16S-23S spacer region rDNA sequences (64 bp) of some cultured species and tobacco chloroplast sequences, where (+) represents positive and (-) negative chains, respectively.

Figures 5A-B show the construction of the border of the vector pSBL-Ct.

Figures 6A-B show the construction of a pSBL-CtV1 selectable vector marker gene cassette containing the 16S chloroplast promoter (Prrn), the aadA gene, and the 3 ′ untranslated region of the psbA chloroplast gene.

Figures 7A-7G show the vectors pSBL-CtV2, pSBL-CtV3, pSBL-CtVH and pSBL-CtVHF, respectively.

Figures 8A-B show the vectors pSBL-CtVHBt and pSBL-CtVHBtR, respectively.

Figure 9 shows transformed and non-transformed tobacco plants growing in the presence of spetinomycin, indicating non-lethal selection on a given medium (500 μg / ml). The growth of transformed (bleached) and non-transformed (green) leaves is noted.

Figures 10A-G show the plastid transformation and regeneration of maize.

Figure 11 shows the plastid transformation of corn. Transformed corn plants grow normally (medium shoot), while non-transformed plants die on a lethal medium, confirming lethal selection with an antibiotic (1000 μg / ml, spectinomycin).

Figures 12A-12E show plastid transformation and regeneration of rice.

Figures 13A and 13B show a PCR analysis of DNA isolated from leaves of rice transformants.

Figure 14 shows the plastid transformation of peanuts. Transformed peanut plants grow normally (middle and left side of the figure), while non-transformed plants die on a lethal medium (500 μg / ml spectinomycin).

Figure 15 shows the plastid transformation of soy. Two transformed plants shoot, while other plants die on a lethal medium, confirming lethal selection with an antibiotic (500 μg / ml, spectinomycin).

Figure 16 shows the transformation of sweet potato embryos on lethal selection medium (500 μg / ml spectinomycin).

Figure 17 shows the transformation of grape cells. Transformed cultured cells turn green, while non-transformed cells die in a lethal selection medium (500 μg / ml spectinomycin).

Figure 18 shows the synthesis of a protein-based biopolymer (PBP) in E. coli. using chloroplast integration and expression vectors.

Figure 19 shows Southern blot analysis performed with transformants of PBP transgenic plants using a tobacco vector (TV). Probes from chloroplast boundary sequences (A) or from polymer gene sequences (EG121) (B).

Figure 20 shows Southern blot analysis performed with transformants of PBP transgenic plants using a universal vector (UV). Probes from the boundary sequences of chloroplasts (A) or from the aadA gene (B).

Figure 21A shows the content of transcripts of a foreign gene analyzed by Northern blotting using complete RNA isolated from a control plant, chloroplast transformants, and a nuclear transgenic tobacco plant, intensively expressing the synthetic biopolymer gene (EG121).

Figure 21B shows an enlarged image of the strips 4-7 from figure 21A.

Figure 22 shows a Western blot analysis of a purified polymer protein from transgenic plants.

Figure 23A shows an increased growth rate of E. coli containing a tobacco vector with the EPSPS gene.

Figure 23B shows an increased growth rate of E. coli containing a universal vector with the EPSPS gene.

Figures 24A-24B show the integration of foreign genes into the plastid genome using PCR using rbcL and aadA primers (A) or 16SRNA and aadA primers (B).

Figures 25A-25B show, by Southern analysis, the integration of the aroA gene into chloroplast and high generation of homoplasmy using the EPSPS probe (A) or the rcbL-orf512 probe. The integration site is shown in figure (B).

Figures 26A and 26B show the generation of seeds collected from control and transformed tobacco plants, respectively, in the presence of selectable markers.

Figures 27A and 27B show transgenic and control tobacco plants sprayed with glyphosate.

Figures 28A and 28B show tobacco plants that are sensitive (control) and resistant (transgenic) to insects.

Figure 29 shows (Western blot analysis) a complete protein isolated from control and transgenic tobacco plants.

The following describes in more detail preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Universal integration and expression vector.

The universal integration and expression vector of the invention is competent for the stable transformation of chloroplasts of a wide variety of plant species. Heterologous DNA coding sequences can be obtained in a cassette for coding a phenotype, such as herbicide resistance, insect resistance, or other traits. The vector also contains a flanking sequence on each side of the DNA coding sequence that is homologous to the spacer sequence of the chloroplast genome, and such a spacer sequence is conserved in the chloroplast genomes of various plants. With this approach, the stable integration of a heterologous gene into the chloroplast genome of a target plant is facilitated by homologous recombination of flanking border sequences with complementary spacer sequences in the chloroplast genome. A universal vector is competent for the transformation of any plant species.

It was found that the spacer region of trnI and trnA is very conserved in many plant species from cyanobacteria to higher plants, such as monocotyledons and dicotyledons. The trnI and trnA genes flank on each side a spacer region called the “spacer 2 ″ or ″ spa 2 ′ ″ region (Figures 4E-4G). Regions on either side of the spacer region also have almost complete homology with corresponding regions in different plant species from cyanobacteria to higher plants, except that higher plants contain two introns in the trnI and trnA genes. Longer border sequences tend to favor more efficient integration of foreign DNA. Therefore, although not mainly, the homology between the species of the trnI and trnA genes, in addition to the homology of the spacer region, contributes to the efficiency of transformation and integration (Figures 4A-4D).

If longer sequences containing non-homologous parts are included in the vector, the non-homologous part of the sequence will be “braided” and “cut out” during the recombination process and will not integrate into the chloroplast target genome.

Various universal vectors can be created with a spacer region. For example, shorter or longer flanking sequences can be linked to parts or to all trnI and trnA genes adjacent to spa 2.

The preferred universal vector contains flanking sequences and an expression cassette containing the following genetic elements for transcription and translation of the DNA coding sequence, arranged in the following order (from the 5 ′ to 3 ′ ends): a 5 ′ flanking sequence, a promoter functional in chloroplast, DNA sequence with the corresponding cloning site (s) for embedding one or more coding sequences for the desired ilotype phenotype molecule of interest and for a selectable marker, a transcription terminator and a part of 3 'flanking sequence. The order of the DNA sequences encoding the desired phenotype and the selectable marker can be changed. Flanking plant DNA sequences can be added to promote sustainable integration. Preferably, the flanking sequence comprises a replication origin (ori) point.

It is especially noted that the highly conserved spacer region is located in an inverted repeat of the chloroplast genome. However, the special location of the spacer in the chloroplast genome is not as important as its high homology with the spacer region of various plants.

In addition, as can be seen from Figures 4E-4G, the spacer 2 (or spa 2) sequence having a length of 64 bp is too short to include the ori of the chloroplast genome that is located before this spacer. If you need to include ori, you must select a longer spacer sequence containing ori, which will include the spacer sequence and an additional sequence in the flanking sequences. This will result in a longer matrix for homologous recombination into the chloroplast genome of the recipient and promotion of homoplasma.

Another preferred vector is a vector in which the flanking sequences contain, except for spacer 2, each part or all of the intergenic spacer region between the tRNA ^Ile and tRNA ^Ala genes of the chloroplast genome (Figures 4A-4D). In addition, flanking sequences may include parts or all of the tRNA ^Ile and tRNA ^Ala genes, respectively. Optionally, the flanking sequences each contain parts, or entire sequences of rRNA 16S and / or 23S genes.

Examples of universal vectors.

A preferred universal vector comprises a DNA sequence that contains the sequence of the spacer region 2 between the highly conserved trnI and trnA genes between the 16S-23S rRNA genes of the chloroplast genome. Preferably, this region contains part or all of the DNA sequence (figures 4E-4G) of the trnI and trnA genes. This region is excised from a selected tobacco-like plant and subcloned into a conventional available plasmid like pUC19, for example, at the PvuII site. An expression cassette is inserted into the plasmid, which contains a selectable marker gene, a 16SrRNA chloroplast promoter, a gene encoding an antibiotic-resistant enzyme similar to the aadA gene encoding aminoglycoside-3′-adenyl transferase, which confers streptomycin and spectinomycin resistance, and 3 untranslated region of the psbA chloroplast gene.

Specifically, when a universal vector with the pSBL-Ct-bor plasmid is created (FIG. 5), a selectable marker gene cassette containing the chloroplast promoter 163 rRNA, the aadA gene encoding aminoglycoside-3′-adenyltransferase conferring streptomycin resistance and spectinomy is inserted into the plasmid or a herbicide resistance gene, and a 3 ′ untranslated region of the psbA chloroplast gene (Figure 6). Then this selectable marker gene cassette is inserted into the universal border in two different directions. In the pSBL-CtV1 vector, a selectable marker gene cassette is inserted into the trnI gene (Figure 6A). In the pSBL-CtV2 vector (Figure 7A), a selectable marker gene cassette is inserted between the trnI and trnA genes in the spacer region in the direction of transcription of 16S rDNA. In the pSBL-CtV2 R vector (map not shown), a selectable marker gene cassette is inserted between the trnI and trnA genes in the spacer region in the opposite direction to 16S rDNA transcription.

The pSBL-CtV2 vector, the preferred variant of the universal vector, contains some interesting genes. For example, the pSBL-CtV3 vector (Figure 7B) contains the mGFP reporter gene encoding a green fluorescence protein isolated from jellyfish. This gene can also be useful for visible selection of transformed plants or in ornamental horticulture, for example in ornamental crops like Christmas trees, or even in lawn grasses that can produce green fluorescence when illuminated with blue light.

The pSBL-CtVH vector (Figure 7B) contains another selectable marker for hydromycin phosphotransferase (the hph gene obtained with the atpB chloroplast promoter), which confers antibiotic resistance to hydromycin. This vector can be used to transform plants that are resistant to other antibiotics, and is especially useful for transforming monocotyledons that are generally resistant to other commonly used antibiotics. This gene may confer additional traits, such as resistance to herbicides, an unintended trait.

The pSBL-CtVH vector (Figure 7) contains the GFP and hph genes, which can be used for lethal or combined selection - lethal and by visible signs.

A tobacco-specific chloroplast vector and a universal chloroplast vector.

The chloroplast vector for tobacco pZS-RD-EPSPS (Figure 2A) (″ TV ″) and the universal vector pSBL-RD-EPSPS (Figure 2B) (″ UV ″ ”) contain both the Prrn promoter (from 16S rRNA), the aadA gene (for selection with spetinomycin), a mutant aroA gene that encodes the EPSPS synthase enzyme (for selection with glyphosate) and the psbA 3 ′ region. The flanking sequences in pZS-RD-EPSPS contain rbcL and orf 512, and in pSBL-RD-EPSPS contain the trnI and trnA genes, which facilitate integration either into a large region of a unique copy (figure 1, arrow ″ TV ″) or in the region of inverted repeats (Figure 1, arrows ″ UV ″) of the chloroplast tobacco genome, respectively.

Glyphosate is an active ingredient in the Monsanto ROUNDUP ™ herbicide and is used as a selectable marker for the herbicidal selection of transgenic plants.

Construction of chloroplast vectors.

Standard vector construction schemes are used, including dephosphorylation of the ends completed by the Maple fragment. Tobacco chloroplast expression vector pZS-RD-EPSPS is shown in Figure 2A. Universal chloroplast vector is shown in figure 2B. The construction of these vectors is also shown in the examples. Both plasmids are amplified in E. coli XL1 Blue strain. Growth curves are recorded in minimal medium M-9. Both vectors are used for glyphosate selection to confer ROUNDUP ™ resistance.

The chloroplast expression vector pZS-RD-EPSPS is specific for tobacco and, as noted earlier, unsuitable for transformation of other plants (Maier et al., 1995). In contrast, the universal chloroplast expression and integration vector pSBL-RD-EPSPS (Figure 2B) is competent to transform the chloroplastic genomes of many other plant species due to the universality of the vector, as described above. A universal vector integrates foreign genes into the spacer region of the 16S-23S chloroplast genome. In the universal vector, trnA and trnI genes (transfer chloroplast RNAs encoding alanine and isoleucine) from the region of the inverted chloroplast genome repeat are used as flanking sequences for homologous recombination. The chloroplast border sequence used in this invention also contains a chloroplast origin of replication (oriA), which is confirmed for some crop species, including peas (Nielsen et al., 1993) and tobacco (Lu et al; 1996), which may explain homology of highly conserved sequences in this field. This point of origin of replication provides an increased number of plasmid matrices for effective integration into the recipient's chloroplast genome and the achievement of homoplasmy.

As shown above, when constructing a universal vector in a suitable restriction site, an expression cassette containing a chloroplast promoter, a selectable marker gene conferring antibiotic resistance (or another selectable marker), a gene encoding a target molecule is inserted into a DNA fragment containing a spacer region and other elements (described here). If necessary, the foreign gene encoding the target molecule can be inserted into the expression cassette after the cassette is inserted into the DNA fragment containing the conserved spacer region, so that before the cassette is inserted, the cassette will contain many cloning sites for the insertion of one or more DNA coding sequences.

The location of the restriction site in the spacer sequence can determine the corresponding length of the two flanking sequences, which will be parts (or different or the same length) of the spacer region. Thus, there is no need for the identity of the two flanking sequences in length, as long as each of them contains sufficient complementarity to the chloroplast target genome to promote homologous recombination.

Since the vector of the invention has homology of such a high degree to the spacer region of the chloroplast genomes of many plant species, it is competent to transform not only the plant species from which the boundary sequence of the vector is derived, but also for any plant species.

As used herein, the term “homologous” means that a DNA sequence from one plant species has sequence regions identical to parts of a DNA sequence from another plant species. In other words, if two DNA sequences are highly homologous, DNA samples may have 100% sequence identity or sequence identity less than 100%. For example, for integration into a chloroplast genome, a 400 bp sequence that has a total homology of only 25% but which contains a 100 bp part homologous to 85-90% or more is considered highly homologous to the chloroplast genome.

It is also shown that the inclusion of a chloroplast ori in the flanking sequences of a universal vector is favorable. Without going into theory, it is believed that the presence of ori in the universal vector promotes homoplasmy after one selection cycle without the need for a second selection stage. This is especially important during the transformation of monocotyledonous plants, such as maize or rice, in which the second selection stage is not feasible due to the difficulties of growing these plants when cultivated from leaf cuttings, as a result of which it is necessary to grow these plants from embryos. If ori is necessary, but lost, it can be entered into the flanking sequence or elsewhere. If it is necessary to increase the number of copies of the introduced universal vector, the chloroplast DNA fragment containing ori should be inserted outside the flanking sequence, so that it will function only to amplify the number of copies of the universal vector and will not integrate into the chloroplast genome.

In contrast to obtaining from a particular plant, flanking sequences can be obtained from the spacer region obtained synthetically, as shown below.

For transcription and translation of a DNA sequence encoding a polypeptide of interest, the entire promoter region of a gene capable of expression in the chloroplast is used, as a rule. The promoter region may include promoters that can be obtained from chloroplast genes, such as the spinach or pea psbA gene, the maize rbcL and maize atpB promoter regions, and rRNA promoters. Competent promoters are also described in patent No. 5693507, which also refers to other literature.

The flanking sequences indicated in patent No. 5693507 and other publications for promoting sustainable integration are not flanking sequences of the universal expression and integration vector described herein and are highly conserved in plants from species to species, while flanking sequences described in this patent and other publications are not.

Identification of intergenic spacer sequences.

The invention relates to methods for identifying the corresponding non-transcribed intergenic spacer sequences in plants that are suitable for creating universal vectors. The method includes isolation of plastid genomic DNA, hybridization with a radioactive isotope-labeled probe of a known spacer, detection and isolation of plastid sequences exhibiting the desired degree of homology with the probe. By way of example, in order to determine whether the plastid gene has an unknown structure and sequence in the spacer region, Southern blotting is carried out using tobacco as a probe in the spacer region. Plastid genomic DNA is isolated and digested with a suitable restriction enzyme in accordance with established procedures. Hybridization with a spacer probe is carried out both under stringent conditions (for example, 50% formamide at 68 ° C, washing in 0.1 × SSC at 68 ° C), and under mild conditions (for example, 6 × SSC at 68 ° C, washing in 2 × SSC at 50 ° C) (1 × SSC is a solution of 0.15 M NaCl, 0.015 M sodium citrate), and plastid sequences exhibiting approximately 90-100% homology or 60-100% homology with the tobacco spacer, respectively. Identified plastid sequences are then isolated. If the requirements for homologous recombination are less stringent, one can be satisfied with a low degree of hybridization with the probe, such as about 60%.

Thus, any known or unknown spacer region with sufficient homology for recombination is suitable for creating UV. Similarly, the known sequence of any intergenic highly conserved spacer sequence can be used to identify isolated plastid sequences that are homologous to the known spacer sequence.

Alternatively, the BLAST program described above can be used to identify highly conserved regions in plastid genomes whose sequences are known.

Plants that can be transformed.

Plants that can be transformed using the universal vector of the invention include any lower plants, such as cyanobacteria, and any higher plants, such as monocotyledonous and dicotyledonous plant species. Transformable plants can be plants growing in sunlight or plants growing underground. A non-limiting list of examples of higher plants that can be transformed using a universal vector includes cereals such as barley, corn, oats, rice and wheat, melons such as cucumber, netted melon and watermelon; legumes such as beans, Chinese vigna, peas, peanuts; oilseeds such as canola and soy; nightshade plants such as tobacco; tuber crops such as potatoes and sweet potatoes; vegetable crops such as tomato, pepper and radish; fruit crops such as pear, grapes, peach, plum, banana, apple and strawberries; fiber crops similar to the genus Gossypium, such as cotton, flax and hemp; and other plants, such as beets, cotton, coffee, radish, industrial flowering plants, such as cloves and roses; cereals, such as sugarcane or lawn grass; evergreen trees such as fir, spruce and pine, and deciduous trees such as maple and oak. Of major interest are the main economically important crops like maize, rice, soy, wheat and cotton. As far as the authors of the invention know, none of these plants, except tobacco, has never undergone a stable transformation using the chloroplast genome, and not one, including tobacco, has undergone a stable transformation using the universal vector described here. The plant from which the DNA sequence is usually derived is tobacco because it is the most fully characterized plant, but the chloroplast genome of any other plant is suitable.

It should be recalled, as described above, that the vector used for the sustainable transformation of tobacco is incompetent for the transformation of the chloroplast genome of other plants.

The method of transformation. Expression cassettes can be transformed into a plant cell of interest in any well-known manner. Such methods include, for example, the following methods: transformation by means of particle bombardment of tungsten, transformation through the mediation of polyethylene glycol, the use of a laser beam, electroporation, microinjection, or any other method that allows the introduction of DNA into the chloroplast. See, for example, Sanford, 1988; Daniell, 1993; Daniell 1997; U.S. Patent No. 5,693,507; Kin Ying et al., 1996. Using these methods allows the application of the invention described herein to be applied to a wide variety of both monocotyledonous and dicotyledonous plants.

Expression of non-plant molecules in transformed plants

The increased utility of the universal expression integration vector of the invention is clearly demonstrated by the competence of the vector for producing transformed plants for the expression of non-plant and valuable molecules.

Protein-based polymers that break down under the influence of microorganisms.

In accordance with another embodiment of the invention, a universal vector is used to transform tobacco with a synthetic gene expressing protein-based polymers (PBP). Such polymers and their expressing genes are known from the literature (Daniell, et al., 1997). Of particular interest are protein-based polymers (PBPs) containing pentameric sequences similar to GVGVP (Yen et al., 1987). Such RTB polymers exhibit useful properties of the inverse phase temperature transition. Protein becomes insoluble when the temperature rises above the transition temperature. PBPs are a wide range of materials similar to petroleum polymers, such as hydrogels, elastomers and plastics. They also show significant biocompatibility, and this makes them suitable for a number of medical applications, including the prevention of postoperative adhesions, tissue reconstruction, and planned drug delivery (Urry et al., 1993). In addition to medicine, possible applications include the use in transducers, molecular devices, superabsorbents and plastics, degradable by microorganisms (Daniell et al., 1997). Such polymers include polymer (Val ¹ -Pro ² -Gly ³ -Val ⁴ -Gly ⁵ ) _n (VPGVP) _n , where n can vary from 1 to hundreds of units, for example 250, or its analogues. Important industrial opportunities and related aspects are examined by Daniell, 1995. Useful plastics that are biodegradable by microorganisms are obtained from the PBP. Genes expressing such PBPs are also useful in the invention in that they can be used as carriers, i.e. the gene of the desired molecule can be hybridized with the gene of the PBP integration and expression of chloroplast.

In previous studies, the synthetic polymer gene encoding (GVGVP) ₁₂₁ was hyperproduced in E. coli to such an extent that inclusion polymer bodies occupied almost 80-90% of the cell volume (Guda et al., 1995; Daniell et al., 1997) . The same gene is also expressed in the nuclear compartment of cultured tobacco cells (Zhang et al., 1995) and in the leaves of transgenic tobacco plants (Zhang et al., 1996).

In the model, the intergenic region of the trnI and trnA genes in the spacer region of the 16S-23S rRNA of the tobacco genome (Figure 1) is used to create a universal vector for integration of the selectable marker gene, aadA gene, and the synthetic polymer gene (EG121). The vector is inserted into the region of the inverted repeat of the chloroplast genome. Transformed tobacco plants express large amounts of protein. Chloroplast genomes of other plant species can also be transformed with a synthetic gene for expression of a protein-based polymer using a universal vector.

Getting high-value molecules. Studies with biopolymers have shown that a non-plant product can be expressed using a synthetic gene, and it is possible using the vectors of the invention to synthesize biologically valuable molecules in transformed plants with a variety of coding DNA sequences. The DNA coding sequence will be contained in a universal vector or, if required, in an expression cassette, as described above.

It is known that transgenic plants produce biologically active molecules by nuclear transformation, rather than chloroplast transformation. See the following links, all of which are incorporated herein by reference. Daniell 1995; Miele 1997; Lyons, 1996; Daniell and Guda, 1997; Arntzen 1997.

Expression of biologically active molecules.

Plants transformed in accordance with the invention with a universal vector or expression cassette can be obtained for the expression of valuable biologically active molecules in the chloroplast-containing parts of the plant. Then the plants are harvested by methods known in practice. Transformed plants containing these products can thus be administered orally. Arntzen, 1997. The production of pharmaceuticals using transgenic plants has been carried out with peptides (proteins) for many pharmaceutical applications, including vaccines, immunomodulators, growth factors, hormones, blood proteins, inhibitors and enzymes. Typical biologics derived from transgenic plants reported in the literature are viral disease vaccines; viral peptide epitopes like human immunodeficiency virus, non-viral peptide epitopes, bacterial antigenic proteins; bioactive peptides, recombinant toxins, plantibodies (recombinant antibodies), serum proteins and secondary plant metabolites. All of these products can be expressed in chloroplasts of transgenic plants in accordance with the invention.

Typical pharmaceutical peptides or proteins produced in transgenic plants are hepatitis B surface antigen, norwalk virus capsid protein, foot and mouth disease virus, human rhinovirus 14, human immunodeficiency virus, S. mutans surface protein, E. coli enterotoxin, B subunit, round malaria epitopes sporozoites, epitope of the mouse protein ZP3 (vaccine); mouse catalytic antibody 6D4, mouse mAB Guy′s 13, mAB B1-8, anti-phytochrome Fv protein, antibody P (antibody); human serum albumin (HSA), human protein C (serum protein); α-trichosanthin, ricin (cytotoxin); human epidermal growth factor (growth factor); leienkephalin (neuropeptide) and acidic human β-glucosidase (hGC) (enzyme). Many of these molecules are expressed in tobacco, potato tubers, and the like.

Of particular interest in accordance with the invention is the production of insulin and human serum albumin (HSA) using a universal integration and expression vector or expression vector. HSA has already been obtained in transgenic (nuclear) potato and tobacco plants (Sijmons et al., 1990). The above products can be obtained through the transformation of chloroplasts in accordance with the invention.

Insulin.

The invention relates to a method for expressing insulin in a transformed plant in the form of a protein fused to a polymer. The plant can be transformed with an expression cassette or a universal integration and expression vector containing a synthetic biopolymer gene like (GVGVP) ₄₀ . Tobacco is a suitable plant because of the ease of processing by genetic engineering methods and the need to find alternative uses for this controversial crop.

In the case of expression in bacteria, the method includes creating a vector for expression in E. coli as a polymer gene (GVGVP) ₄₀ fused to the proinsulin gene, expression of polymer-insulin fusion proteins in E. coli (grown in a known manner), purification of the recombinant protein using thermal transition properties of a protein-based polymer; and cleavage of insulin from the polymer using well-known methods.

An expression cassette or universal integration and expression vector containing a polymer gene fused to the proinsulin gene is introduced to transform a plant into a target plant like tobacco. The polymer-insulin fusion protein is expressed in the plant, the protein fused to the polymer is extracted from the chloroplast and cytosolic compartments of plant cells, the insulin is cleaved from the polymer-protein using ditreitol (DTT) or other known methods and collect insulin. Alternatively, the fusion product of insulin and polymer can be expressed in a food culture or in the edible parts of a crop.

The method of fusion of a DNA sequence encoding a molecule with biological activity with a synthetic gene expressing a protein-based polymer for expression in a suitable bacterial host, or yeast, or in a transformed plant is a very promising method for widespread use.

Recombinant human serum albumin in plants. Recombinant human serum albumin (rHSA), indistinguishable from human authentic protein, was obtained in nuclear transgenic plants of tobacco and potato (Sijmons et al., 1990). This event showed the expression of valuable protein in transgenic plants, but also that appropriate treatment can be achieved by fusion of HSA with the plant sequence, which was manifested in cleavage and secretion of the desired protein. The chloroplast genome of a selected tobacco-like plant can be easily transformed with the universal vector described here and induce HSA expression.

General applicability.

As described herein, a universal vector allows for plant transformation in accordance with the invention, induces the plant to express a biological molecule that can confer the desired phenotype to the plant, and / or produce the desired product that may, but need not, possess biological activity (or the precursor of the final product). The coding nucleotide sequence may be synthetic or natural. The produced molecule may be alien to plants, not functioning in the plant or functioning. Universal vector has a wide range of applications in the field of plant transformation.

It is assumed that any biologically active molecule (its precursor or derivative) can be obtained using a transgenic plant transformed with the universal vector of the invention, with appropriate adaptation, which may be required in a particular case.

Tolerance to herbicides transformed in plant chloroplasts

Another embodiment of the invention relates to the use of a universal vector for imparting herbicide tolerance or tolerance to plants. Advances in gene transfer technology have made it possible to introduce a herbicide resistance gene into plants, whereby herbicidal selectivity for a given crop is achieved.

Modifications of the target enzyme. The first stage in this approach is the identification and necessary modification of the target enzyme (gene) of the herbicide to give tolerance, which is carried out quite extensively. Sulfometuron-methyl is a sulfonylurea class herbicide that blocks the growth of bacteria, yeast and higher plants by inhibiting the first enzyme of the branched chain of amino acid reactions of acetolactate synthase (ALS). Mutant genes for ALS that confer resistance to sulfometuron methyl are isolated from E. coli and yeast (Yadav, et al., 1986). Herbicide resistance of tobacco and some other crops was achieved by genetic engineering methods with the ALS gene (Gabard, et al., 1989; Miki, et al., 1990). Another approach to creating herbicide resistance in plants is the expression of the phosphinotricin acetyltransferase (PAT) enzyme, which detoxifies the phosphinotricin herbicide (DeBlock, et al., 1987).

Glyphosate.

Glyphosate is a powerful broad-spectrum herbicide, highly effective against annual and perennial cereal and broad-leaved weeds. Glyphosate is safe for the environment, as it decomposes quickly in the soil, has minimal mobility in the soil and has very low toxicity to non-plant life forms. Glyphosate acts by binding and inhibiting the enzyme 5-enol-pyruvyl shikimate-3-phosphate (EPSP) synthase. EPSP synthase (EPSPS) catalyzes the formation of EPSP, which is important in the biosynthesis of aromatic amino acids from shikimat-3-phosphate and inorganic phosphate. Glyphosate is non-toxic to animals due to the fact that this reaction occurs only in plants and microorganisms. Unfortunately, since the EPSP formation reaction occurs in all plants, glyphosate does not make a choice between weeds and useful plants, such as agricultural and ornamental crops.

Two approaches can be used when trying to develop a glyphosate resistant plant by genetic engineering. One approach is based on the overproduction of wild-type ESPS synthase, so that after competing inhibition of ESPS synthase by glyphosate, the remaining ESPS synthase confers glyphosate resistance. Another approach is based on the expression of a mutant gene (aroA) encoding glyphosate-resistant ESPS synthase.

In all, without exception, the above examples, herbicide resistance genes were introduced into the nuclear genome.

The need for chloroplast transformation.

There is a serious need to develop a herbicide-resistant plant, especially a plant that is resistant to most commonly used herbicides, in which a herbicide-tolerant protein is produced in chloroplast, from which the herbicide-tolerant gene cannot be released into the environment with pollen. .

The universal vector of the invention meets this need by transforming any target plant with resistance to any selected herbicide like glyphosate. Crops important to the economy, like wheat, rice, corn (maize), soybeans, can be made resistant to the selected herbicide using a universal vector.

The invention relates to a transgenic herbicide resistant plant in which a foreign transgene conferring resistance to one or more herbicides is integrated into the chloroplast genome using a universal vector. The transgenic plant may be a mature plant, an immature plant, such as a seedling, germ, callus, cultured tissue or cell suspension, or a part of the plant, such as a stalk or callus. Herbicides suitable for the invention, in which genes that confer resistance, can be stably integrated into the chlorollast genome in accordance with the invention, include all known (as well as developed) herbicides.

Class of herbicides.

Herbicides that are susceptible to the effects of the method of the invention are generally divided into several chemical classes.

The first class includes PSII herbicides (photosystem II), which prevent the restoration of plastoquinone at the PSII acceptor center; it includes chemicals such as triazines, triazinones, urea derivatives, carbamates, nitriles, nitrophenols, substituted pyridazinones, phenylcarbamates, anilides cyanoacrylate, DCMU, carboanilides, uracils, specifically, for example, dichlorophenyl dimethyl urea, atrazine, metribuzin, lenacyl, phenmedipham, ioxinyl and dinoseb. These chemicals bind to the 32 kD binding protein (Q _B protein, D1, herbicide binding protein) in the thylakoid membrane of chloroplasts, whereby they block electron transfer during photosynthesis. The plastid gene encoding the Q _b protein precursor is called psbA, and its sequences exhibit a very high degree of homology in various plants. The most important PSII class herbicide is atrazine, developed by Ciba-Gergy.

Another class is the PSI herbicides (photosystems I), the membrane-bound protein complex of which catalyzes light-controlled oxidation of plastocyanin (PC) and the reduction of ferrodoxin (Fd). Typical chemicals are bipyridyl paraquat and diquat herbicides.

Another class of herbicides is aryloxyphenoxypropanoates (APP) or herbicides such as acetyl coenzyme A carboxylase, which inhibits acetyl CoA carboxylase and subsequent biosynthesis of fatty acids in plastids. Typical APPs are cidohexanedione (CHD), setoxidine, haloxifol, hesalofop, phenoxaprop (and its lower alkyl substituted molecules), dicolofop, setoxidine, celodine, and tralcoxydim.

Another class of herbicides are auxin analogues, such as macropop, chloramben, dicamba, benazolin, 1-naphthylacetic acid; 3,6-dichloropicolinic acid, picloram, fluoroxypyr, hinklorac, MCPA and 2,4-D.

Another class is mitototic herbicides called dinitroaniline herbicides, similar to trifluralin, oryzaline and pendimethalin.

Another chemical class of herbicides to which the invention is applicable are herbicides active in the biosynthesis of amino acids, such as tertiary aminomethylphosphonic acids, chlorosulfuron, glufosin and glyphosate.

Another class of herbicides is acetolactate synthase (ALS) inhibiting herbicides like sulfonylureas, imidazolinones, triazolopyrimidines and pyrimidinylthiobenzoates, such as chlorosulfuron, imazafir, flumettsulam (and others listed in Section 1, Table 1, Table 1, Resistance 1994, cited below).

Examples of sulfonylurea herbicides are sulfometuron methyl (active ingredient Oust ™) and chlorosulfuron (active ingredient Glean ™). One of the imidazolinones imazapyr is an active ingredient in the American Cyan-amid Arsenal ™ herbicide, and imazametabenz is a mixture of two imidazolinones (Merk Index, 11th Ed. 4825). Mutated forms of ALS localized in the ALS ilvG and ILV2 structural genes can be used to confer herbicide resistance using a universal vector.

Despite the chemical differences between imidazolinones and sulfonylureas, these substances inhibit the same ALS enzyme. It turns out that quinones and an imidazolinone herbicide compete with a sulfonylurea herbicide for a common center on ALS. Accordingly, according to the invention, plants can be transformed in such a way that they will be resistant to both groups of those and other herbicides.

A typical representative of another group of chemicals that can be controlled by the invention using a universal vector and having herbicidal activity is L-phosphino-tricin, which is a component of the bialaphos tripeptide. This tripeptide is sold under the trade name ″ Herbias ™ ″ by Meiji Seika, Japan, and as ″ Basta ™ ″ Hoechst AG, Germany. L-Phosphinotricin is a potent inhibitor of glutamine synthetase, causing a rapid increase in the concentration of ammonia in plants, and leads to the death of plant cells.

Despite extensive research, a satisfactory product to give plants tolerance to herbicides through the transformation of chloroplasts has not been developed. In the case of a glyphosate-type herbicide, regenerated transgenic plants transformed in the nucleus show glyphosate tolerance, but also show growth degradation and do not lead to transgenic plants with a high level of glyphosate tolerance (Shultz, et al., 1990).

Chloroplast transformants.

In accordance with the invention, the chloroplast of herbicide-sensitive target plants is transformed with a vector of the invention bearing the desired coding sequence, whereby resistance to herbicides is imparted. The transformed chloroplast contains a genome that carries a foreign transgene that confers resistance to herbicides. The chloroplast may be a mature chloroplast or may be an immature chloroplast, such as an etioplast. Preferably, the herbicide resistance gene encodes an EPSP synthase that binds less readily (has reduced affinity) to glyphosate than wild-type EPSP synthase. If another transgene is present, it is usually a gene that gives the plant resistance to antibiotics. Thus, herbicide resistance can be used as a marker for lethal selection in the case of chloroplast transformation by selecting transformants when exposed to a medium with a lethal concentration of the selected herbicide in which the transformants will survive.

The source of the second gene may be prokaryotic (e.g., bacterial) or eukaryotic (e.g., plant).

The invention also relates to the preparation of a plant resistant to several herbicides, whether they are herbicides of the same class or herbicides of different classes, and a transformed plant resistant to many herbicides.

It is known that some plant species have developed a natural and temporary tolerance to certain types of herbicides. The provisions of this invention are easily applicable to them.

The invention relates to a method for producing a herbicide-resistant plant, comprising transforming a plant chloroplast by introducing one or more foreign transgenes encoding a herbicidal resistance protein into the plant chloroplast genome. Preferably, the transgene encodes a mutant form of an enzyme having a lower affinity for this herbicide compared to a naturally occurring enzyme.

Table I below lists the different types of resistance determinants (chemicals or ″ molecules ″) that inhibit or confer resistance, and typical herbicides corresponding to them.

For a comprehensive review of plant resistance to herbicides, see ″ Herbicide Resistance Crops, Agricultural, Environmental, Economic, Regulatory and Technical Aspects ″, 1996, CRC Press, Inc., Editor, Stephen O. Duke, and ″ Herbicide Resistance in Plants, Biology and Biochemistry ″, 1994, CRC, Press, Edited by Stephen B. Powels and Joseph AM Hoi turn. In the first of these books, of particular interest is Chapter 3, devoted to methods for producing sustainable crops (and the numerous references cited in it), as a prerequisite for the creation of the invention. Both books are incorporated herein by reference.

In accordance with the invention, it was also found that in the process of expressing the intended trait, the transformed plant may express another trait that may be very necessary, may even be more necessary than the originally intended trait. In such a situation, expression can be allowed in plants and selection based on this other trait.

The invention is further described by way of non-limiting examples.

Example 1

Universal chloroplast integration and expression vectors

Tobacco. Exemplary universal chloroplast vectors are created by first cutting out the BamHI tobacco chloroplast DNA fragment (130656-140992) containing the rRNA 16S and 23S genes and subcloning it into the usual available bacterial plasmid pUC19. A map of the chloroplast genome of tobacco is shown in Figure 1. The 2.1 kb HindIII-EcoRI fragment in this fragment containing the universal border sequence containing the trnI and trnA genes (Figure 5A), including the spacer region between these genes, is subcloned into the plasmid pUC19 at the PvuII site (Figure 5B). The resulting plasmid is designated pSBL-Ct Bor (Figure 5B).

The pSBL-RD-EPSPS vector (Figure 2B) contains a mutant EPSP synthase gene encoding an EPSP synthase enzyme. Glyphosate The active ingredient in the ROUNDUP ™ herbicide, Monsanto, binds to the EPSP synthase protein and blocks the synthesis of essential amino acids, resulting in plant death. The EPSP synthase encoded by the mutant gene does not bind glyphosate and thus gives the crop plants resistance to the herbicide.

Other genes, such as genes that confer resistance to environmental hazards, such as salt and drought tolerance (osmotolerance genes such as betainaldehyde dehydrogenase (BADH), for excessive glycine betaine production), or thermal tolerance (genes encoding heat shock proteins) , or cold shock resistance or pathogen resistance proteins, such as antimicrobial (lytic peptides, chitinase) or antiviral (envelope proteins), can be administered individually or in non-conflict combinations in a universal a vector for chloroplasts or in different cassettes of the same universal vector for chloroplasts, for transforming the target plant into a plant with the desired trait.

Design of a universal chloroplast integration vector containing a synthetic spacer region 2

A universal vector for chloroplasts containing only the spacer region 2 of the tobacco chloroplast genome is constructed by first subcloning a synthetic oligonucleotide containing the spacer region 2 into the bacterial plasmid pUC19. Synthesize the plus and minus chains of the spacer sequence in 64 base pairs, where the sequence of the plus chain is as follows:

5′-GCTGCGCCAGGGAAAAGAATAGAAGAAGCATCTGACTACTTCATGCATGCTCCACTTGGCTCGG-3 ′.

Synthetic fragments are mixed and annealed, and then ligated into pUC19 at the PvuII site (Figure 5B). The insertion of the corresponding selectable marker gene and the homologous gene is carried out as described above for pSBL-CtBor (Figure 5B).

In order to obtain a longer sequence including the tRNA ^Ile and tRNA ^Ala genes, the same methodology is used.

Transformation of various plants

Example 2

Transformation of tobacco chloroplasts.

This example describes a classic tobacco chloroplast transformation scheme for which any vector can be used. Two such vectors are identified below. All new vectors for chloroplasts are first tested on tobacco, as described in Daniell (1997). Tobacco plants (Nicotiana tabacum, cultivar Petit Havana) are grown aseptically by seed germination on MSO medium containing MS salts (4.3 g / l), a mixture with vitamin B5 (myoinositol, 100 mg / l; thiamine-Hcl, 10 mg / l; nicotinic acid, 1 mg / l; pyridoxine-HCl, 1 mg / l), sucrose (30 g / l) and phytagar (6 g / l), at pH 5.8. For the bombardment, fully bloomed leaves of plants at the age of about two months are selected.

The leaves for the bombardment are placed abaxially on Whatman No. 1 filter paper lying on RMOP ^* medium in standard Petri dishes (100 × 15 mm). The desired plasmid DNA (for example, pSBL-RD-EPSPS or pZS-RD-EPSPS) is applied to tungsten (1 μm) or gold (0.6 μm) microparticles and bombardment is carried out using a PDS1000 / He biolistic device (Bio-Rad) as described in Daniell, 1997. After the bombardment, Petri dishes were sealed with parafilm and incubated at 24 ° C. under a 12-hour light period. Two days after the bombardment, the leaves are cut into small pieces of approximately 5 mm ² and placed on a medium for lethal selection (RMOP containing a selectable marker, such as about 500 μg / ml spetinomycin dihydrochloride), in deep (100 × 25 mm) Petri dishes (approximately 10 pieces per cup) so that the abaxial side touches the medium. Separated from dead shoots, regenerated spectinomycin-resistant shoots are cut into small pieces (about 2 mm ² ) and subcloned into clean deep Petri dishes (about 5 pieces per cup) containing the same lethal selection medium. Resistant shoots of the second culture cycle are transferred to growth medium (MSO medium supplemented with IBA, 1 μg / L, and an appropriate antibiotic, for example 500 μg / ml spectinomycin dihydrochloride). The plants that have taken root are transferred to the soil and grown at 26 ° C under continuous light for subsequent analysis.

After transfer to the lethal selection medium, the explants gradually turn pale, and after about 3-8 weeks, green calluses and shoots develop from the bombarded side of the 0 leaf. Sustainable shoots from each callus are considered as a clone.

PCR screening of chloroplast transformants after the first cultivation cycle showed that in 12 of the 20 resistant clones, foreign genes like the aadA gene linked to the EG121 gene were integrated into the chloroplast gene. These 12 clones are passed on to the next stages of regeneration. The entire regeneration process, from bombardment to transfer to soil, takes about 3-5 months.

Figure 9 shows transformed and non-transformed tobacco plastids growing in the presence of spectinomycin, which indicates non-lethal selection on this medium (500 μg / ml).

Example 3

Transformation of corn chloroplasts.

Surface sterilization and germination of corn seeds. Corn seeds are subjected to surface sterilization in a solution containing 20% (v / v) industrial bleach and 0.5% SDS for 15 minutes under shaking conditions, then washed four to five times in succession in sterile double-distilled water (sddw). Germination fluid (modified with CGS) containing MS salts (4.3 g / l), sucrose (30 g / l), DM vitamins (1.0 g / l thiamine-Hcl, 0.5 mg / l nicotinic acids, 0.5 mg / L pyridoxine-Hcl and 100 mg / L myoinositol) and BA (2.0 mg / L) at pH 5.8 are dispensed in a Magenta ™ chamber (45 ml) containing eight layers of hard gauze, and then autoclave. Seeds (25 seeds of any genotype per chamber) are placed in a modified CGS medium and cultivated for three days (16 hours of continuous illumination; 25 ° C) for germination. Nodal sections are aseptically excised from three-day plants. The nodal section looks like a clear demarcation on a developing seedling and represents the seventh node (Figure 10A). Excised nodal sections are approximately 1.2-1.5 mm long (Figure 10B).

Figures 10A-G show the transformation of corn plastids and a regeneration scheme. A) Three-day seedlings of corn; arrows and line show the seventh node for excision of the explant; B) nodal cross sections before the bombing; arrows indicate the edge of one slice; C) GUS-positive nodal section (core transformation); histochemical analysis performed three days after the bombardment; D) the induction of numerous shoots from one nodal cut (control) after eight weeks of cultivation; D) a control shoot on the medium for elongation after three weeks; E) rooted control seedling; G) selection of corn transformed in plastids in a liquid medium containing spectinomycin and streptomycin for eight weeks.

Induction of numerous shoots. Nodal section explants are placed on corn shoot induction medium (CSI; MS salts, sucrose and DM vitamins as described above, BA (2.0 mg / L), CPA (0.25 mg / L) and phytagar (8 g / l) at pH 5.8) with an acropetal end up and placed in the conditions for cultivation indicated above. In all media except modified CSG and RG1 (Figure), PGR and antibiotics are sterilized by filtration and added after autoclaving. Tissues are subcultured every two weeks on fresh CSI medium to form numerous shoots (Figure 10E). The subordinate shoots are separated from the shoot group after eight weeks of cultivation and elongated on MS semi-solid medium containing sucrose, DM vitamins, glycine (10 mg / L) and asparagine (150 mg / L), at pH 5.8 over three weeks (Figure 10D). Sprouts take root (Figure 10E) on the same medium containing IBA (0.5 mg / L). Sprouts with roots can be grown on MS liquid without PGR in test tubes (150 × 25 mm) containing harsh gauze as a retaining material to achieve faster growth. The regenerated sprouts are transplanted into the medium for growing in pots, allowed to acclimatize, and then grown to a mature state in the greenhouse.

Figure 11 shows the plastid transformation of corn. Transformed corn plants grow normally (medium shoot), while non-transformed plants die on a lethal medium, which confirms lethal selection with the help of the spectinomycin antibiotic (1000 mg / ml).

Example 4

Transformation of rice chloroplasts.

Surface sterilization of rice seeds and pre-cultivation. The husked seeds of any genotype (Indian or Japanese varieties) are subjected to surface sterilization, first in 70% ethanol for 10 minutes under continuous shaking, then washed with ddw about five times. Then the seeds are immersed in a 0.2% (m / v) solution of benlat for 20 minutes, washed with sddw five times, then immersed in 50% bleach for 20 minutes, and washed again with sddw. Seeds were pre-cultured in RG1 medium (MS salts, sucrose and DM vitamins, as described above, BA (0.2 mg / L) at pH 5.8). As with corn, liquid RG1 is dispensed before autoclaving in Magenta chambers containing harsh gauze. Seeds are placed in RG1 (100 seeds of any genotype per chamber) and pre-cultured overnight (16-hour or continuous illumination; 23 ° C) before being bombarded the next day.

Embryo bombardment on intact rice seeds.

Pre-cultivated seeds are tightly laid vertically with the germinal end up (Figure 12A) in the central part with a diameter of 2.5 cm Petri dish (25 per cup) containing Rg1.1 medium (RG1 with the addition of 8.0 g / l phytagar), and bombarded with coated DNA microparticles.

DNA precipitation.

The procedure is the same as described for corn, with the following changes. Use 10 μl of DNA (1.0 μg / ml) and 20 μl of isopropanol (2X DNA volume), 60 μl of a 2.5 M CaCl ₂ solution and 15 μl of a 0.1 M spermidine solution. Each dose delivers 2.0 μg of DNA and 720 μg of tungsten.

Figures 12A-E show plastid transformation of rice and regeneration scheme. A) Rice seeds, germinal end up, immediately before the bombing; arrows indicate the edges of the embryo; B) the induction of numerous shoots from one control embryo after seven weeks of cultivation; C) selection of rice shoots transformed in plastids emerging from one initial rice germ in a medium containing spectinomycin and streptomycin after eight weeks on selective medium; arrows indicate two putative transformants; D) control restored rice plants; E) transgenic Priscilla 2.3; E) transgenic Priscilla 2.4.

Induction of numerous shoots and selection of plastid transformants after the bombardment. Rice seeds are separated and sown on RG1.1 medium (preserving polarity) and after the bombardment they are placed in the dark for two days. Then the germinal end of the seed is cut off (the germ with a small amount of endosperm) from the rest of the endosperm, which is discarded. The embryos are placed in 50 ml of RG2 liquid medium (in a 250 ml flask) to induce numerous shoots (Figure 12B). RG2 contains MS salts, sucrose and DM vitamins, as described above, and BA (6.0 mg / L) at pH 5.8. The RG2 used for the selection contains spectinomycin (1000 μg / ml) supplemented with streptomycin sulfate (100 μg / ml). The cultures are placed in a growth chamber (16-hour light period; 25 ° C) and subcultured every two weeks in fresh medium for selection. From the group of shoots that emerged from each embryo, green shoots are selected and again placed in a selective medium (Figure 12B). Root appearances are achieved in RG3 medium (MS salts, sucrose and DM vitamins, as described above, IBA (0.5 mg / L, pH 5.8) with the addition of antibiotics. (Roots may appear either individually or as groups of roots.) Sprouts are transplanted into the medium for growing in pots, allowed to acclimatize, then again transplanted into pots with a mixture of clay and sand (1: 1) and grown to a mature state in the greenhouse (figures 12G, D, E).

Development of plastid transformation and regeneration schemes for corn and rice.

As described above, unique schemes for the transformation of corn kernels and regeneration (Figure 10) have been developed (Rudraswamy, 1997) and adapted to transform plastids. (Prior to this work, nodal slices explants were not used for transformation or regeneration.) Numerous shoots are induced on nodal slices excised from three-day-old seedlings of 21 genotypes (none of them are related to A188 or B73), which include hybrid (16-grain, one sweet) or inbred (four) genotypes. After eight weeks of cultivation, 16-32 (average 24) shoots per explant are generated. The stems are rooted, and the restored plants do not show aberrant phenotypes in analyzes in the greenhouse (studies were limited to two plants per genotype). DNA can also be delivered to the explants of nodal sections of all genotypes (Figure 10B; unstable expression of β-glucuronidase). For plastid transformation, nodal section explants bombard pSBL-ctV2, then they are placed on a medium for the induction of numerous shoots containing spectinomycin and streptomycin. Emerging shoots can be cut out and placed again on the medium for induction for subsequent selection cycles.

As described above, the unique husked intact mature rice target seeds, with the germinal end up, not used in the transformation schemes previously reported, are combined with the induction scheme of numerous shoots (Figure 12) for mature embryos (excised two days after the bombardment). Numerous shoots are induced on all eight tested genotypes (Litton and Priscilla - two new varieties bred in the Mississippi, plus six bred lines). The noted reaction should be similar in many other varieties, since the initial explant is a mature embryo. Recovered (non-transformed) plants are stored to collect F1 seeds. After plastid transformation, the propagation of shoots occurs in the presence of spectinomycin / streptomycin, and, as in the case of corn, shoots can be subjected to many selection cycles due to the proliferation of shoots (it is not known whether they are axillary or accessory by origin) from the base of excised shoots. Rooting is also carried out on selective media.

Figures 13A-B show a PCR analysis of DNA isolated from leaves of the first generation of rice transformants. PCR products are not as abundant as observed in chloroplast transgenic tobacco plants (Figure 13A, lane 11, Figure 13B, lane 12). There are two reasons for this. The scheme used to isolate DNA is not suitable for large rice leaves, or primers designed for tobacco do not renature just as well with ct of rice DNA. However, tobacco primers are used for preliminary analysis to verify the integration of the aadA gene into the plant genome from a universal vector. The lack of product may indicate spontaneous mutants capable of growing on spectinomycin without the aadA gene (Figure 13A, lanes 7-10). A 1.57 kb PCR product was detected in four lines (Figure 13A, lanes 2-6) transformed with a universal vector. Under the selection conditions used, four mutants from ten lines transformed by a universal vector were found. Primers are also created to specifically identify integration into the plastid genome. In the case of a universal vector, the primer on the native chloroplast genome is placed in the 16S rRNA gene outside the flanking sequence of the chloroplast vector (1.60 kb PCR product). The expected products are observed in the case of transgenic lines obtained using a universal vector (Figure 13B, lanes 5, 6). As expected, no PCR products were detected in plants not subjected to bombardment (control) (Figure 13B, lane 2; Figure 13A, lane 1). PCR results reveal two 'Priscilla' rice plants (2.3 and 2.4) containing transformed plastids (figures 12 and 13).

Example 5

Transformation of Peanut Chloroplasts (Arachis hypogaea)

Transgenic peanuts with transformed chloroplast genomes are prepared using the universal vector pSBL-CG-CtV2 (Figure 7A). Peanut tissue is grown under cultivation in accordance with the scheme described by Kanyand et al., 1994. The bombardment conditions are the same as in the case of the transformation of tobacco chloroplasts described above, except that slices of epicotyl are used for bombardment despite the use of variable pressure bursting discs . The transformation of peanut chloroplasts has never been reported before.

Figure 14 shows the plastid transformation of peanuts. Transformed peanut plants grow normally (in the middle and on the left side of the figure), while non-transformed plants die on a lethal medium (500 μg / ml).

Example 6

Transformation of soy chloroplasts.

Transgenic soybeans containing transformed chloroplast genomes are prepared using the universal vector pSBL-CG-CtV2 (Figure 7A). The bombardment conditions are the same as in the transformation of tobacco chloroplasts. Transformation of soy chloroplasts has never been reported before.

Figure 15 shows the plastid transformation of soy. Two transformed plants show shoots, another plant dies on a lethal medium, confirming lethal selection with the help of the spectinomycin antibiotic (500 μg / ml).

Example 7

Transformation of sweet potato chloroplasts.

Transgenic sweet potato plants containing transformed chloroplast genomes are prepared using the universal vector pSBL-CG-CtV2 (Figure 7A). The sweet potato tissue is grown during cultivation in accordance with the scheme described by Zhang et al., 1996. The conditions for bombardment are the same as for the transformation of tobacco chloroplasts described above, except that calluses and primary embryos are bombarded and transferred to cups after bombardment containing 100 mg / ml spectinomycin. Transformation of sweet potato chloroplasts has never been reported.

Figure 16 shows the transformation of sweet potato embryos on a lethal selective medium with spectinomycin antibiotic (500 μg / ml). Bleached calluses (right) and green buds (left) are noted.

Example 8

Transformation of grape chloroplasts.

Transgenic grape plants containing transformed chloroplast genomes are prepared using the same universal vector pSBL-CG-CtV2. The grape tissue is grown during cultivation in accordance with the scheme of Hebert et al., 1993. All chloroplast transformation schemes are the same as in the case of tobacco, except that cells are used for bombardment in the exponential growth phase approximately 4 days after subcloning. The transformation of grape chloroplasts has never been reported before.

Figure 17 shows the transformation of grape cells. Transformed culture cells turn green, while non-transformed cells die in a lethal selective medium with the spectinomycin antibiotic (500 μg / ml).

Example 9

Transformation of other plants.

Microparticle bombardment of plants is an advantageous method of introducing a universal vector into target plants that carries the desired nucleotide sequence encoding a molecule of interest. Typical examples of transgenic plants obtained by microparticle bombardment are shown in table. II.

The transformation of plants using a gene gun is described by Daniell, 1997. Each culture reported that it can be transformed into a nucleus by microparticle bombardment, according to this table, can have its own chloroplast genome transformed using the universal vector described here.

Example 10

Expression of non-plant products

The following examples illustrate the expression of microbial degradable protein-based biopolymers (PBPs) and analysis of transformants.

Vector pSBL-CG-EG121.

The pSBL-CG-EG121 vector (FIG. 3A) contains a 121mer gene (GVGVP) ₁₂₁ (called EG121) that encodes a microorganism-degradable protein-based biopolymer (PBP) having numerous medical and non-medical uses.

Construction of chloroplast expression vectors. Conventional vectoring schemes are as described by Sambrook et al., 1989. The chloroplast integration and expression vectors pSBL-CtV2 (Figure 7A) and pZS197 cleave XbaI (a unique site between the aadA gene and the psbA 3 ′ region) and SpeI (a unique site through 120 bp after the aadA gene in the regulatory region of psbA 3 ′), the sticky ends are completed using the Klenov fragment and dephosphorylated. The EG121 polymer gene together with the Shine-Dalgarno sequence (GAAGGAG) from the pET11d vector is excised from the pET11d-EG121 plasmid as an XbaI-BamHI fragment. The sticky ends of the inserted fragment are completed using the Klenov fragment and ligated with the vector pSBL-CtV2 or pZS197, resulting in the production of chloroplast expression vectors pSBL-CG-EG121 (Figure 3A) and pZS197-EG121 (Figure 3B), which provide integration of the aadA and EG121 to the inverted repeat (IR) or to the spacer region between the rbc1 and orf 512 genes of the chloroplast tobacco genome. See Figure 1, UV and TV integration sites, respectively.

Expression of biopolymers in E. coli and tobacco.

The plasmid vector pSBL-CG-EG121 (Figure 3A) was used to transform E. coli XL-1 Blue strain and grown in terrific broth in the presence of ampicillin (100 μg / ml) at 37 ° C for 24 hours. SDS-PAGE electrophoresis carried out according to Laemmli, 1970, using a 12% separating gel and 5% concentrating gel for 5 hours at a constant current of 30 mA. Coarse crude protein extracts were prepared from E. coli cells and subjected to electrophoresis as described in Guda et al., 1995. After electrophoresis, the polypeptides were visualized by negative staining with CuCl ₂ .

Figure 18 shows the expression of chloroplast integration and expression vectors in E. coli strain HMS174 (DE3). Lane 1 corresponds to a purified polymer protein; lane 2 corresponds to the control of untransformed E. coli; lane 3 corresponds to E. coli strain XL-1 Blue transformed with the universal vector pSBL-CG-EG121 (Figure 3A); lane 4 corresponds to E. coli transformed with the tobacco vector, and lane 5 corresponds to E. coli strain HMS174 (DE3) transformed with the pET11d-EG121 vector in which the T7 promoter transcribes the polymer gene. The expression level using the Prrn promoter in E. coli is almost equivalent to that obtained using the highly potent T7 promoter that controls the polymer gene.

Southern blotting.

Total DNA from the leaves of transformed plants and wild-type plants was extracted using the CAB method of Rogers and Bendich, 1988.

Figures 19 and 20 show Southern blotting performed independently with transformants obtained using the tobacco vector (Figure 19) and the universal vector (Figure 20). Total DNA is digested with EcoRI and HindIII in the case of universal vector (UV) transformants or EcoRI and EcoRV in the case of tobacco vector (TV) transformants. The presence of an EcoRI site at the 3 ′ end of the polymer gene allows fragments of the predicted size to be cut out only in chloroplast transformants. In order to confirm the integration of a foreign gene and homoplasmy, individual blots are analyzed using probes corresponding to the boundary sequences. In the case of TV transformants, after the second or third selection cycles, the boundary sequence hybridizes with 4.6 and 1.6 kb fragments (Figure 19A, bands 2, 3, and 4) and a native 3.1 kb fragment in the wild type (Figure 19A lane 1). On the other hand, in the case of UV transformants, after the first selection cycle, the boundary sequence hybridizes to 4.0 kb and 1.2 kb fragments (Figure 20A, bands 1 and 2), while in the control it hybridizes to the native fragment in 2.1 kb (Figure 20A, lane 3). Moreover, a 3.1 kb native fragment (Figure 19A, bands 2 and 3) is also found in the TV transformants, similar to a wild-type plant, which indicates the heteroplasmic state of the transformed chloroplast genomes, even if they have passed several selection cycles. At the same time, both UV transformants show a homoplasmic state even after the first selection cycle (Figure 20A, lanes 1, 2).

The presence of heteroplasmy after the second selection was previously reported, and it is assumed that selection must be performed before homoplasmy is achieved (Svab and Maliga, 1993). This is consistent with the observation that TV transformants show a high degree of heteroplasmy after the second selection cycle (Figure 19A, bands 2 and 3). At the same time, in the case of UV transformants, no heteroplasmic state was found, which may be due to the mechanism of copy correction between the two IR regions and / or the presence of a chloroplast origin of replication (ori) within the boundary sequence, which can increase the number of copies of the introduced plasmid before integration.

DNA blots also analyze either the aadA gene (plants with UV integration) or the EG121 gene (plants with TV integration) to reaffirm the integration of foreign genes into chloroplast genomes. In plants with integrated TV, the polymer gene probe hybridizes with a 4.6 kb fragment only in plastid transformant lines (Figure 19B, bands 2, 3, and 4). Also in plants with integrated UV, the aadA sequence hybridizes to a putative 4.0 kb fragment (Figure 20B), which also hybridizes to the boundary sequence in plastid transformant lines (Figure 20A, lanes 1 and 2).

Analysis of the content of transcripts in transgenic tobacco and Northern blotting.

The content of transcripts of a foreign gene is analyzed by Northern blotting (Figure 21) using total RNA isolated from a control plant, chloroplast transformants and a transgenic tobacco plant expressing the polymer gene (EG121) with high levels through the nuclear genome (Zhang et al., 1996 ) The polymer gene sequence (EG121) hybridizes with a 1.8 kb fragment in chloroplast transformants (bands 1-4 and 5-6), as well as with larger fragments in one of the chloroplast transformants (lane 6). In the case of a nuclear transform, a transcript of approximately 2.1 kb is observed (lane 7). This is due to the presence of the poly-A sequence at the 3′-end of the polymer transcript provided by the nos terminator. The larger fragments observed in band 6 may be di-, tri- or polycistronic transcripts that are processed in chloroplasts. This is a common occurrence in gene expression in chloroplasts, since many plastid genes assemble into polycistronic transcription units, which leads to the appearance of complex sets of overlapping mRNAs. A quantitative determination of the content of transcripts shows that chloroplast transformants produce 11 times (lane 5) or fifty times (lane 6) more polymer transcripts than the nuclear transformant with a high level of expression (lane 7, the plant with the highest expression among thirty-five TV-checked nuclear transgenic plants). This is directly related to the presence of a greater number of gene copies in the chloroplasts of transformed plants. Plastid transformants with integrated tobacco vector (TV) show a lower polymer transcript content (lanes 1-4) compared to transformants with integrated universal vector (lanes 5, 6), because the polymer gene exists in two copies per transformed plastid gene in transformants universal vector versus one copy in TV transforms, and due to heteroplasmic states observed in TV transforms.

Western blotting.

The polymer protein is isolated in pure form from wild-type tobacco leaves, chloroplast transformants and nuclear transgenic plants in accordance with the method recently described by Zhang et al., 1995. The purified polymer is analyzed by SDS-PAGE electrophoresis according to Laemmli, 1970, using 12% separating gel and 5% concentrating gel for 5 hours at a constant current of 30 mA. Approximately 60 kD polypeptides were visualized by negative staining with 0.3 M CuCl ₂ . The gels decolorize in a 0.25 M solution of Na-EDTA and 0.25 M Tris-Cl, pH 9.0, with three buffer changes at intervals of 10 minutes. Western immunoblotting and staining (Figure 22) is carried out as described in Zhang et al., 1996, using a monoclonal antiserum against the AVGVP polymer, which crosshairs interacts with the GVGVP polymer, and the Immuno-Blot Assay Kit (Bio-Rad ) Polypeptides with a mobility of approximately 60 kD are found in plastid transformants integrated into IR plants. The expression of the polymer by a high expression level F2 generation nuclear transgenic plant (the plant with the highest expression among 35 transgenic plants was tested) is visible in lane 5 (Figure 22), while no polymer is expressed in non-transformed control plants, as seen in lane 4 (figure 22). In chloroplast transformants, the level of expression of polymer transcripts is found to be eleven to fifty times higher (Figure 21). In the case of native proteins that are found in chloroplasts, such as valine and proline, whose biosynthesis pathways are comparamentalized in chloroplasts, a higher level of protein production can be expected.

Example 11

Genetic engineering for glyphosate tolerance through the nuclear and chloroplast genomes

Chloroplast integration and expression tobacco vectors containing EPSPS.

The EPSPS coding sequence has recently been integrated into the chloroplast genome of tobacco (Figure 1). The chloroplast vector pZS-RD-EPSPS (Figure 2A) contains the 16S rRNA (Prrn) promoter, which controls the aadA and EPSPS genes, with the psbA 3 ′ region from the tobacco chloroplast genome. This design allows integration of the aadA and EPSPS genes into the spacer region between the rdcL and orf512 genes of the tobacco chlorollast genome. Figure 1, arrow ″ TV ″.

Glyphosate resistance test.

Gene expression in E. coli. Due to the high similarity of transcription and translation systems in E. coli and chloroplasts (Brixey et al., 1997), chloroplast expression vectors were the first to be tested in E. coli for resistance, in this case to glyphosate, before transforming higher plants. The higher growth rate of E. coli containing the tobacco vector compared to the control containing pZS197 (similar to pZS-RD-EPSPS but lacking the EPSPS gene) in the presence of 10 mM and 40 mM glyphosate (Figure 23A) indicates tolerance to glyphosate of E. coli expressing the EPSPS gene. Another growth curve (Figure 23B) confirms the expression of EPSPS in E. coli using a universal vector. Thus, the resistance of E. coli to glyphosate is a consequence of the expression of the EPSPS gene present in both tobacco and universal vectors.

Characterization of transgenic tobacco plants

Gene integration.

The fully bloomed leaves of Nicotiana tabaccum, a variety of Petit Havana, are bombarded with tobacco and universal chloroplast vectors. Two days after the bombardment, leaf explants are transferred to lethal selection medium containing spectinomycin (500 μg / ml). Transgenic plants receive within 3-5 months after the bombardment. As a rule, of the 16 bombarded leaves, 10 independently transformed shoots are identified.

PCR analysis is carried out with DNA isolated from shoots of the first or second generation, as well as from mature transgenic plants. To confirm the integration of the aadA gene into the plant genome, primers based on tobacco and universal vectors are used. Lack of product may indicate spontaneous mutants capable of growth on spectinomycin without the aadA gene. The expected PCR product (887 bp) was detected in six lines (Figures 24A-B, lanes 1-6) transformed with the tobacco vector. In four lines transformed with a universal vector, a PCR product of 1.57 kb was detected (Figures 24A-B, lanes 1-4). Under the used selection conditions, four mutants were detected from ten lines transformed by the tobacco vector. On the other hand, integration of the aadA gene was found in all transgenic lines transformed by a universal vector.

PCR to confirm integration into chloroplasts.

Primers have also been created to specifically identify integration into the plastid genome. Here, the strategy is to make one primer correspond to the native chloroplast genome near the site of integration of the vector, while the other corresponds to the aadA gene. A primer was created that is located immediately immediately after the rbcL gene of the tobacco vector (2.08 kb PCR product). In the case of a universal vector, the primer is localized in the native chloroplast genome in the 16S rRNA gene (1.60 kb PCR product). Expression products were detected in the case of transgenic lines obtained using the tobacco vector (figures 24A-B, lanes 2-7), as well as using the universal vector (figures 24A-B, lanes 1-4). In plants not subjected to bombardment (control), as expected, PCR products were not detected (Figure 24A, lanes 1 and 9; Figure 24B, lanes 5 and 11). Thus, it turns out that all tested plants are chloroplast and not nuclear transformants, probably due to the requirement for a higher content of aminoglycoside adenyl transferase (AADA) in transgenic plants under strict selection conditions. Low levels of AADA in nuclear transgenic plants should lead to the removal of nuclear transformants. The results of PCR analysis are convincing and prove the integration of foreign genes into chloroplasts using both tobacco and universal vectors.

Southern analysis.

The integration of the aroA gene into chloroplasts is also confirmed by Southern analysis. In addition, the observed high level of glyphosate resistance (Figure 20A) is confirmed by determining the number of copies of a foreign gene in transgenic plants. The probe for determining the integration of a foreign gene into the chloroplast genome is a 654 bp EPSPS gene fragment labeled with P ³² using the scattered seed method. Total DNA, including both DNA from organelles and from the nucleus, is digested with EcoRI. Availability of EcoRI site for 200 bp in front of the integration site in the chloroplast genome is used to confirm the integration of the EPSPS gene into the chloroplast genome of transgenic plants. Upon hybridization of the probe with the native EPSPS gene present in the nuclear genome, a 4.5 kb fragment is detected. In addition, when hybridizing the probe with the split chloroplast genomes of transgenic tobacco plants, fragments of 3.5 and 4.35 kb are detected, figure 25A, bands 2, 3 and 4. The probe does not hybridize with the split chloroplast gene of the untransformed control plant (figure 25A, lane 1), since the foreign gene is not present in the chloroplast genome of tobacco. This clearly confirms the integration of the EPSPS gene into the chloroplast genome.

The number of copies of genes.

The number of copies of the integrated gene is determined by establishing homoplasmy of the transgenic chloroplast genome. Tobacco chloroplasts contain 5,000 to 10,000 copies of their genome per cell (McBride et al., 1995). If only a part of the genomes is actually transformed, the number of copies should be less than 10,000. Having established that only the genome transformed by EPSPS is present in transgenic plants, it can be established that the number of copies is 5000-10000 per cell. This is confirmed by cleavage of total EcoRI DNA and hybridization with flanking sequences capable of homologous recombination into the chloroplast genome. The probe is a 2.9 kb fragment of rbcL-orf 512 sequences. The chloroplast genome transformed with the EPSPS gene contains the EcoRI site in the rbcL-orf 512 region of the chloroplast genome, resulting in the formation of an additional fragment upon cleavage by this enzyme (Figure 25B). When analyzing by Southern hybridization in the case of a non-transformed control plant, a 4.43 kb fragment is detected, figure 25B, lane 1. Two fragments (4.35 kb and 3 kb) are formed in lanes 2, 3 and 4 due to the inclusion of the EPSPS gene regions rbcL and orf512 (Figure 25B is a diagram with dark dashed lines showing the place of integration of foreign DNA). The 4.43 kb fragment available in the control is absent in transgenic plants. This proves that only the transgenic chloroplast genome is present in the cell and that there is no native non-transformed chloroplast genome without the EPSPS gene. This indicates the homoplasmic nature of the transformants, and at the same time an estimate of the number of copies is given - 5000-10000 copies of the foreign EPSPS gene per molecule. This may explain the high level of glyphosate tolerance observed in transgenic tobacco plants (Figure 20A).

Progeny.

Seeds collected from self-pollinated transgenic plants are germinated in the presence of spectinomycin (500 μg / ml). All seeds germinate, remain green and grow normally (Figure 26B). Uniform resistance to spectinomycin shows that the aadA gene is transmitted to all offspring. The absence of color suggests homoplasmy, since a heteroplasmic state could lead to multi-colored offspring on spectinomycin (Svab et al., 1990; Svab and Maliga, 1993). The absence of variations in chlorophyll pigmentation in the offspring also emphasizes the lack of a positional effect of nuclear transformation. All control seedlings are discolored and do not grow in the presence of spectinomycin (Figure 26A).

Glyphosate tolerance. Eighteen-week-old control and transgenic plants are sprayed with equal volumes of glyphosate at different concentrations (0.5-5 mM). Control tobacco plants are very sensitive to glyphosate; they die within seven days even at a glyphosate concentration of 0.5 mM (Figure 27B). On the other hand, chloroplast transgenic plants survive at such high glyphosate concentrations as 5 mM (Figure 27A). These results are interesting, given the fact that the EPSPS gene from petunia used in these chloroplast vectors has a low glyphosate tolerance and also contains a transition peptide for directing to chloroplasts.

This description is the first report on eukaryotic nuclear gene expression within the prokaryotic chloroplast compartment. It is well known that the frequency of codon usage varies significantly between the prokaryotic chloroplast compartment and the eukaryotic nuclear compartment. Ideally, the mutant aroA gene (which does not bind to glyphosate) from the prokaryotic system should be expressed in the chloroplast compartment. Now, such genes are available and show a level of glyphosate resistance thousands of times higher than the petunia gene used in this work. In light of these observations, it is possible that the integration of prokaryotic herbicide resistance genes into the chloroplast genome, carried out here, can lead to an incredibly high level of resistance to herbicides, while biological retention efficiency, i.e. pollen dispersion is avoided.

Example 12

Glyphosate tolerance of corn.

A universal chloroplast vector using corn chloroplast DNA is constructed as follows. First, the pSBL-Ct-bor vector is constructed (Figure 5B). Based on the bacterial plasmid pUC19, a subclone of maize chloroplast DNA is created containing one of the regions of inverted repeat. Then, a smaller subclone containing only the rRNA operon is created from the first subclone, and a fragment present in the second subclone containing the trnA and trnI genes and spacer regions representing the universal boundary are subcloned into the pUC19 plasmid at the PvuII site. The resulting plasmid was named pSBL-Ct-bor. In the pSBL-Ct-bor plasmid, to create the pSBL-CORN vector, a selectable marker gene cassette containing the 16S rRNA chloroplast promoter, the aadA gene (encoding aminoglycoside-3′-adenyltransferase conferring resistance to streptomycin, spectinomycin) and 3 ′ untranslated are inserted psbA. A selectable marker gene cassette is inserted between the trnI and trnA genes in the spacer region in the direction of 16S rDNA transcription.

The pSBL-CORN-aroA vector containing the aroA mutant gene from Salmonella typhimurium (Stalker et al., 1985; Comai et al., 1983), which encodes the EPSPS synthase enzyme, is created by inserting the aroA mutant gene into the pSBL-CORN vector. Transgenic maize plants expressing the aroA mutant gene are resistant to glyphosate treatment like ″ Roundup ™ ″, while non-transformed control plants are not resistant.

Example 13

Chloroplast transformation for tolerance to imidazolinones or sulfonylureas.

The plasmid pSBL-CORN is modified by inserting a DNA fragment containing the mutant form of the Saccharomyces cerevisiae acetolactate synthase gene (Faico and Dumas, 1985; Yadav et al., 1986) to obtain the plasmid pSBL-CORN-ASLI. This gene encodes acetolactate synthase, which is not inhibited by imidazolinones or sulfonylureas and which confers resistance to herbicides containing sulfomethuron-methyl and herbicides containing imazapyr. Transformed tobacco plants in which the mutant gene for acetolactate synthase is expressed are resistant to spray herbicides based on imidazolinones and sulfonylureas.

The pSBL-CORN-ASL2 vector is a derivative of pSBL-CORN that contains mutated copies of the suRA and suRB tobacco genes (Chaleff and Ray, 1984). These genes encode a tobacco acetolactate synthase polypeptide. Plasmid pSBL-CORN-ASL2 is created by ligation of the suRA and suRB genes isolated from tobacco genomic DNA with the pSBL-CORN vector. The resulting vector confers resistance to imidazolinone and sulfonylurea herbicides.

Example 14

Chloroplast transformation for tolerance to photosystem II inhibitors.

The resistance of the photosystem (PS) II to herbicides is determined by mutations in the psbA gene encoding the Q _B protein and is very conservative in all plants. Resistant plants possess mutations that alter amino acids at specific specific positions of Q _B proteins, such as residues 219, 251, 255, 264, and 275 (Hirschberg and McIntosh, 1993; Galloway and Mets, 1994; Gloden and Haselkorn, 1985; Erickson et al. 1984; Johanningmeier et al., 1987). Genes possessing such mutations can thus be used to confer resistance to herbicides whose action is to inhibit electron transfer by the PS II system.

Examples of such herbicides are dichlorophenyl dimethyl urea (DSMU), atrazine, metribuzin, lenacyl, phenmedifam, loxinyl and dinoseb.

A mutant psbA gene containing the substitution of serine for glycine at residue 264 is isolated from the Chlamydomonas genomic DNA using appropriate restriction endonucleases. The resulting fragment can be ligated with the universal chloroplast expression vector pSBL-ctV2 and transformed into E. coli XL1Blue. A purified plasmid from this E. coli strain is used to transform plants (Daniell, 1997). The introduction of mutant psbA genes into the chloroplast genome and the selection of the corresponding transformants is carried out as described previously. Transformed plants producing a mutant psbA protein containing serine to glycine substitution are resistant to atrazine ™, while control plants are unstable.

A mutant psbA gene containing the substitution of valine for isoleucine at residue 219 was isolated from the Chlamydomonas genomic DNA using appropriate restriction endonucleases. A universal vector is constructed as described above. Transgenic maize-like plants expressing psbA containing valine to isoleucine substitution at residue 219 are expected to be resistant to spraying with DCMU sprays.

Example 15

Tolerance to auxin analogues, such as 2,4-P.

The universal chloroplast expression vector pSBL-ctV2 can be cleaved with XbaI and ligated with a DNA fragment containing a gene encoding a monooxygenase. The resulting construct can be transformed into chloroplasts to generate transgenic plants containing multiple copies of the monooxygenase gene. It is expected that the resulting plants expressing a large amount of monooxygenase will be tolerant to 2,4-D.

Example 16

Chloroplast transformation for insect resistance.

Tobacco plants can be transformed with the universal vector pSBL-CtVHBt (Figure 8A), which contains the cryIIA gene and expresses the CryIIA protoxin, thereby conferring resistance to insect pests similar to the Pyralidoe family, such as Brazhnik. Even insects that have developed resistance or are less sensitive to Bt toxin are killed by the Bt toxin expressed by the gene in the chloroplast vector described here.

The pSBL-CtVHBt vector is created by digesting pSBL-CtVH SmaI and ligating the product with the cryIIA gene encoding the CryIIA protein. The product contains the cryIIA gene in the correct transcriptional orientation (Figure 8A).

The integration of the cryIIA gene into the chloroplast genome of tobacco is confirmed by PCR. The number of copies of cryIIA per cell is estimated to be 10,000 by Southern blotting. CryIIA protein, as estimated by Western blotting, accounts for 5 to 10% of the total amount of cellular protein. This is the highest CryIIA protein content ever reported in the literature for Bt transgenic plants. Bioassays of excised leaves are carried out for untransformed tobacco ″ Petit Havana ″ and transformed cryIIA tobacco. Five to ten caterpillars are placed on each leaf and mortality and damage to the leaf are assessed after 3-5 days. When using sensitive N. virescens (YDK) on transformed cryII-A tobacco, all the caterpillars die within 3 days, while on the untransformed Pet Petit Havana ’, mortality is absent and essentially 100% foliage loss occurs. Similar results are obtained using resistant to CryIAc (YHD2, 40-50000-fold resistance) and resistant to CryII-A (CXC, 2000-fold resistance) N. virescens. In addition, 100% mortality of Helicoverpa zea (cotton scoop) and Spodoptera exigua (small scoop) are observed, none of which has been previously reported that it dies from the Cry protein.

Figures 28A and 28B show a bioanalysis of (A) a control non-transformed plant and (B) a transgenic plant. The tested insects show 100% mortality: they are a scoop sensitive to CryI, a cotton scoop and a scoop small, resistant to CryI and CryII.

Figure 29 shows the total protein isolated by Western blotting from control (lane C) and transgenic plants (lane B). Lanes D-H show different concentrations of purified CryII protein (1-20%). Lane A conforms to protein standards.

Other susceptible insects are described in the description of the invention previously.

As will be obvious to specialists in this field of technology, in the light of the above description, many modifications, changes and replacements are possible in the practical application of the invention without departing from its essence and scope. It is understood that such modifications, changes and substitutions are included in the scope of the claims.

All references cited in this text are without fail included in the present description as references.

Sources of information

Armtzen Ph.D., Charles J. (1997) Public Health Reports 112: 190-197.

Brixey, P. J., Guda, H. and Daniell, H. (1997) Biotechnol. Lett. 19, 395-399.

Carlson, P.S. (1973) Proc. Natl. Acad. Sci. USA 70: 598-602.

Chaleff and Ray. (1984) Science 223: 1148.

Comai, L., Faciotti, D., Hiatt, W., Thomson, G., Rose, R., and Stalker. D. (1983) Science 221: 370.

Daniell, H., Guda, C., McPherson, D.T., Xu, J., Zhang, X. and Urry, D.W. (1997) Meth. Mol. Biol., 63: 359-371.

Daniell, H., and Guda, C. (1997) Chemistry and. Industry, pages 555-558.

Daniell, H., Krishnan, M. and McFadden, B.A. (1991) Plant Cell Rep. 9: 615-619.

Daniell, H., and McFadden. (1987) Proc. Nat. Acad. Sci. (USA) 84: 6349-6353.

Daniell, H., Vivekananda, J., Neilson, B., Ye, G.N., Tewari, K.K., and Sanford, J.C. (1990) Proc. Nat. Acad. Sci. (USA) 87: 88-92.

Daniell H. Porobo Dessai, A., Prakash, C.S. and Moar, W.J. (1994) NATO Asi Series. Ed., J.H. Cherry H86: 598-604.

Darkocsik, C., Donovan, W.P. and Jany, C.S. (1990) Mol. Microbiol. 4: 2087-2094.

Daniell, H., (1995) Inform. 6: 1365-1370.

Daniell, H., Ramanujan, P., Krishnan, M., Gnanam, A. and Rebeiz, C.A. (1983) Biochem. Biophys. Res. Comun 111: 740-749.

Daniell, H. and Rebeiz, S. A. (1982) Biochem. Biophys. Res. Comun. 106: 466-471.

Daniell, H. (1993) Methods in Enzymology. 217: 536-556.

Daniell, H. (1997a) Meth. Mol. Biol. 62: 453-488.

DeBlock, M., Botterman, J., Vandewiele. M., Docky, J., Thuen, C., Gossele, V., Movva, N.R., Thomson, C., Van Montagu, M., and Leemans, J. (1987) EMBO J. 6: 2513-2518.

Erickson et al. (1984) Proc. Nat. Acad. Sci. (USA) 81: 3617.

Falco and Dumas. (1985) Genetics 109: 21.

Gabard, J. M., Charest. P.J., Iyer, V.N. and Miki, B. L. (1989) Plant Phys. 91: 574-580.

Galloway and mets. (1984) Plant Physiol. 74: 469.

Gloden and Haselkorn. (1985) Science 229: 1104.

Guda, S., Zhang, X., McPherson, D.T., Xu. J., Cherry, J., Urry, D.W. and Daniell, H. (1995) Biotechnol. Lett. 17: 745-750.

Hirschberg and McIntosh. (1993) .Science 222: 1346.

Johanningmeier et al. (1987) FEBS Lett 211: 221.

Kanyand et al. (1994) Plant Cell Reports 14: 1-5.

Kin Ying et al. (1996) The Plant Journal 10: 737-743.

King, J. (1996) Science 274: 180-181.

Laemmli, U.K. (1970) Nature 227: 680-685.

Langevin, S. A., Clay. K. and Grace, J.B. (1990) Evolution 44: 1000-1008.

Lewellyn and Fitt (1996) Molecular Breeding 2: 157-166.

Lu, Z., Kunnimalaiyaan, M. and Nielsen, B.L. (1996) Plant Mol. Biol. 32: 693-706.

Lyons, P. C., May, G. D., Mason, H. S., and Arntzen. C. J., (1996) Pharmaceutical News 3: 7-12.

, H. (1995) J. Mol. Biol. 251:614-628.Maier, RM, Necketman, K., Igloi. GL and

, H. (1995) J. Mol. Biol. 251: 614-628.

May, G. D., Mason, H. S., Lyons, P. C. (1996) American Chemical Society, pp. 194-204.

Miele, L. (1997) Elsevier Trends Journals, Vol. fifteen.

R.B. (1996) Nature 380:31.Mikkelson, TR, Anderson, B. and

RB (1996) Nature 380: 31.

Miki, B.I., Labbe, H., Hatori, J., Ouellet, T., Gabard, J., Sunohara, G., Charest, P.J. and Iyer, V.N. (1990) Theoretical Applied Genetics 80: 449-458.

McBride, K.E., Svab, Z., Schaaf, D.J., Hogan, P.S., Stalker, D.M. and Maliga, P. (1995) Bio / Technology 13: 362-365.

Nielsen, B.L., Lu, Z. and Tewari, K.K. (1993) Plasmid 30: 197-211.

Oard, J.H., Linscombe, S.D., Braveramn, M.P., Jodari, F., Blouin. D.C., Leech, M., Kohli, A., Vain, P. Cooley, J.C. and Christou, P. (1996) Mol. Breed 2: 259-368.

Penazloza, V., et al. (1995) Plant Cell Reports 14: 482-487.

Rudraswamy, V. and Reichert, N.A. (1997) M.S. Thesis. Mississippi State Univ.

Rogers. S.O. and Bendich, A.J. (1988) in Plant Molecular Biology Manual, ed. Gelvin. S.B. and Schilperoot, R.A. (Kulwer Academic Publishers, Dordrecht, Netherlands) pp. A6: 1-10.

Sambrook, J., Fritch, E.F. and Maniatis, T. (1989) in Molecular cloning. Cold Spring Harbor Press, Cold Spring Harbor, Mew York.

Sanford, J.C. (1988) Trends In Biotech. 6: 299-302.

Sankula, S., Braverman, M.P., Jordari, F., Linscombe, S.D. and Oard, J.A. (1996) Weed Technol. 11: 70-75.

Schultz, A., Wengenmayer, F. and Goodman, H. (1990) Critical Review in Plant Sciences 9: 1-15.

Shaner and Anderson, P.C., (1985), Biotechnology in Plant Science, 287.

Sijmons, P.C., Cekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., van den Elzen, P.J.M., Hoekema, A. (1990) Biotechnology 8: 217-221.

Stalker, et al. (1985) J. Biol. Chem. 260: 4724.

Stummann et al. (1988) Physiologia Plantarum 72: 139-146.

Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. (USA) 90: 913-917.

Svab, Z., Hajdukiewicz, P. and Maglia, P. (1990) Proc. Mat. Acad. Sci. (USA) 87: 8526-8530.

Umbeck, P.F., et al. (1991) Econ. Entomology 84: 1943-1950.

Urry, D.W., Nicol, A., Gowda, D.C., Hoban, L.D., McKee. A., Williams, T., Olsen, D.B. and Sokh, V.A. (1993) in Biotechnological Polymers: Medical. Pharmaceutical and Industrial Applications, ed. Gebelein, C.G. (Technomic Publishing Co., Inc., Atlanta, GA), pp. 82-103.

Widner, W.R. and Whiteley, H.R. (1989) J. Bacteriol. 171: 961-974.

Yadav, et al. (1986) Proc. Nat1. Acad. (USA) 83: 4418-4422.

Ye, G.N .. Daniell. H. and Sanford, J.C. (1990) Plant Mol. Biol. 15: 809-819.

Yeh, H., Ornstein-Goldstein, N; Indik, Z., Sheppard, P., Anderson, N., Rosenbloom, J., Cicilia, G., Yoon, K. and Rosenbloom, J. (1987) Collagen Related Res. 7: 235-247.

Zhang, X., Guda, S., Datta ,. R., Dute, R., Urry, D.W. and Daniell, H. (1996) Plant Cell Reports 15: 381-385.

Zhang, X., Guda., C., Datta, R., Dute, R., Urry, D.W., Danell, H. (1995) Biotechnology Letters 17: 1279-1284.

Zhang, X., Urry, D.W. and Daniell, H. (1996) Plant Cell Rep. 16: 174-179.

Current Protocols in Molecular Biology. Asubel et al., Eds., John Wiley and Sons, Inc. (1997) Vol. I, II and III.

Herbicide Resistance Crops, Agricultural, Environmental, Economic, Regulatory and Technical Aspects, Duke, S.O., edt., CRC Press, Inc. (1996).

Herbicide Resistance in Plants. Biolocry and Biochemistry, Powles. S. B., and Holtum, J. A. M.; eds., CRC, Press, Inc. (1994).

Claims (29)

1. A universal integration and expression vector competent for the stable transformation of the chloroplast genome of various plant species, including an expression cassette that includes a heterologous sequence encoding a peptide of interest, functionally linked at the 5'- and 3'-end with regulatory sequences that allow expression of the coding sequences in the chloroplasts of the target plant, and flanking sequences located on each side of the expression cassette, omologichnye spacer sequence of the target chloroplast genome, which provide stable integration of the heterologous coding sequence into the chloroplast genome by homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome, wherein the spacer sequence is transcriptionally active and is conserved in chloroplast genome of different plant species. 2. The vector according to claim 1, comprising a heterologous nucleotide sequence that encodes a selectable phenotype. 3. The vector according to claim 2, in which each flanking sequence includes a part of the intergenic spacer region 2 between the tRNA ^Ile and tRNA ^Ala genes of the chloroplast genome, which facilitates double homologous recombination with the conserved spacer region 2 in the chloroplast target genome. 4. The vector according to claim 3, in which each flanking sequence includes, in addition to part of the spacer region, parts or complete genes of tRNA ^Ile and tRNA ^Ala, respectively. 5. The vector according to claim 4, in which each flanking sequence includes, in addition to part of the spacer region, parts or complete sequences of 16S and / or 23SrRNA genes. 6. The vector according to claim 3, in which the spacer region is localized in an inverted repeat of the chloroplast genome. 7. The vector according to claim 3, in which the DNA of the flanking sequences comes from a different plant species than the target plant. 8. The vector according to claim 3, in which the peptide of interest is a biologically active molecule. 9. A method for stable transformation of a target plant, comprising introducing an integration and expression universal vector into the chloroplast genome of the target plant and growing a transformed plant, the vector being competent for stable transformation of the chloroplast of various plant species and includes an expression cassette that includes a heterologous sequence encoding representing interest peptide, functionally linked at the 5'- and 3'-end with regulatory sequences, providing their expression of the coding sequence in the chloroplasts of the target plant, a heterologous nucleotide sequence encoding a selectable phenotype, and flanking sequences located on each side of the expression cassette, including, each, part of the intergenic spacer region 2 between the tRNA ^Ile and tRNA ^Ala genes of the chloroplast genome homologous to the spacer sequence of the chloroplast genome of the target, conservative in the chloroplast genome of various plant species, providing stable integration of goethe ologichnoy coding sequence in the chloroplast genome of the target plant by homologous recombination of the flanking sequences with the homologous sequences in the target chloroplast genome. 10. The method according to claim 9, in which the transformable plant is a monocotyledonous plant. 11. The method according to claim 10, in which the transformed plant is selected from monocotyledonous plants, which are maize, rice, cereal grass, rye, barley, oats or wheat. 12. The method according to claim 9, in which the transformable plant is a dicotyledonous plant. 13. The method according to item 12, in which the transformed plant is selected from dicotyledonous plants, which are soy, peanuts, grapes, sweet potatoes, peas, canola, tobacco, tomato or cotton. 14. The method of claim 9, wherein the peptide of interest is a protein-based synthetic polymer. 15. The method of claim 14, wherein the protein-based synthetic polymer contains repeating pentameric sequences (GVGVP) _n , where n is an integer from 1 to 250, G is glycine, V is valine, and P is proline. 16. The method of claim 14, wherein the expressed peptide of interest is insulin. 17. The method of claim 16, further comprising isolating insulin. 18. The method according to clause 16, in which the insulin is expressed in the form of proinsulin. 19. The method of claim 18, wherein the proinsulin is fused to a protein-based synthetic polymer. 20. The method according to clause 16, in which the transformed plant is tobacco. 21. The method of claim 14, wherein the peptide of interest is a human serum albumin polypeptide. 22. The method according to item 21, in which the transformed plant is tobacco. 23. A method of imparting herbicide resistance to a target plant, comprising introducing into the plant a universal integration and expression vector competent for stable transformation of the chloroplast of various plant species, including an expression cassette that includes a heterologous sequence encoding a protein of interest to the herbicide that is resistant to the herbicide associated at the 5'- and 3'-end with regulatory sequences that provide expression of the coding sequence in chloroplasts of the target plant, a heterologous nucleotide sequence encoding a selectable phenotype, other than tolerance to the indicated herbicide, and flanking sequences located on each side of the expression cassette, including, each, part of the intergenic spacer region 2 between the tRNA ^Ile and tRNA ^Ala genes of the chloroplast genome, homologous spacer sequence of the chloroplast genome of the target, conservative in the chloroplast genome of various plant species, ensuring stable hetero integration the coding sequence into the chloroplast genome of the target plant by homologously recombining flanking sequences with homologous sequences in the chloroplast target genome, and growing the transformed plant. 24. The method according to item 23, in which the protein of interest is an enzyme, and the DNA sequence encoding this enzyme encodes a mutant form of the enzyme having a lower affinity for the herbicide than the natural enzyme. 25. The method according to item 23, in which the enzyme is an EPSP synthase, and the herbicide is glyphosate. 26. The method according to item 23 for the stable transformation of the phenotype of the target plant, in which a breeding phenotype and another trait is expressed in the grown transformed plant in addition to expressing said phenotype. 27. The method according to p. 26, in which the selected phenotype is provided by expression of the hydromycin-β-phosphotransferase gene, and an additional feature is the herbicide glyphosate resistance. 28. A method for determining chloroplast transformation and expression of an intended trait for acquiring a target plant resistance to a selected herbicide due to plant transformation, comprising introducing into the plant a universal integration and expression vector competent for stable chloroplast transformation of various plant species, including an expression cassette that includes a heterologous sequence encoding the desired intended trait functionally linked at the 5'- and 3'-end to sequences providing expression of the coding sequence in the chloroplasts of the target plant, a heterologous nucleotide sequence encoding a selectable phenotype, and flanking sequences located on each side of the expression cassette, including each part of the intergenic spacer region 2 between the tRNA ^Ile and tRNA ^Ala genes of the chloroplast genome, homologous to the spacer sequence of the chloroplast genome of the target, conservative in the chloroplast genome of various plant species, about ensuring stable integration of the heterologous coding sequence into the chloroplast genome of the target plant by homologous recombination of flanking sequences with homologous sequences in the chloroplast genome of the target, exposure of the plants into which the vector is introduced, lethal concentration of the herbicide, and selection of plants that do not die under such exposure thereby select transformed plants expressing the intended trait. 29. A stably transformed transcriptionally / translationally active chloroplast genome of a target plant competent to stably integrate a heterologous sequence, including an expression cassette that includes a heterologous DNA sequence of interest encoding a heterologous molecule of interest expressed under the control of regulatory sequences in the chloroplasts of the target plant , and sequentially plant DNA flanking each side of expression to assets that ensure stable integration of DNA into the target chloroplast genome by homologous recombination, the sequence being transcriptionally active and conserved in the chloroplast genome of various plant species, and this DNA is inherited through organelle replication in daughter cells.

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