MXPA00008805A - Novel plant plastid promoter sequence - Google Patents

Abstract

A novel promoter isolated from the 5'flanking region upstream of the coding sequence of the i(Arabidopsis) plastid i(clpP) gene is described. Also described are a novel method for utilizing protein-coding regions of plastid genes to isolate intervening regulatory sequences and a novel method for improving plastid transformation efficiency using exogenous plastid promoters that differ in nucleotide sequence from native plastid promoters.

Description

NOVEDOSA PROMOTING SEQUENCE OF PLANT PLASTIDES The present invention belongs in general to the molecular biology of plants, and more particularly, belongs to a novel plastid promoter isolated from Arabidopsis thaliana, and to methods for its use. The present invention also pertains to a novel method for using protein coding regions of plastid genes in order to isolate intervening regulatory sequences. The present invention further pertains to the use of novel plastid promoter sequences to improve the efficiency of plastid transformation. The transformation of plastids, where the genes are inserted through homologous recombination in all the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous number of copies on the genes of nuclear expression to allow expression levels that can exceed 10 percent of the total soluble plant protein. In addition, "the transformation of the plastid is desirable because the traits encoded by the plastid are not transmissible by pollen, thus obviating the potential risks of an inadvertent escape of the * transgene to the wild congeners of the transgenic plants. Other advantages of the transformation of plastids include the feasibility of the simultaneous expression of multiple genes as a polycistronic unit, and the elimination of the effects of position and silencing of the gene that can be followed by nuclear transformation. plastids are extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, 5,545,818, and 5,576,198, in International Application Number WO 95/16783, and in Boynton et al., Methods in Enzymology 217: 510-536 (1993). , Svab et al, Proc. Nati, Acad. Sci. USA 90: 913-917 (1993), and McBride et al. , Proc. Nati. Acad. Sci. USA 91: 7301-7305 (1994); all of which are incorporated herein by reference. The basic technique for the transformation of the tobacco plastid involves the bombardment with particles of the tissue of the leaf with regions of AD? of the cloned plastid 'flanking a selectable marker, such as an antibiotic resistance gene. Flanking regions of 1 to 1.5 kb, referred to as targeting sequences, facilitate homologous recombination with the plastid genome, and therefore allow the replacement or modification of specific regions of the 156 kb tobacco plastome. Initially, point mutations were used in the AR? R 16S of the chloroplast and in the rpsl2 genes that confer, resistance to spectinomycin and / or streptomycin as selectable markers for the transformation (Svab et al., Proc. Nati. Acad. Sci. USA 87: 8526-8530 (1990), Staub, JM and Maliga, P., Plant Cell 4: 39-45 (1992), both incorporated herein by reference). This resulted in stable homoplastic transformations at a frequency of about one per 100 white bombardments. The presence of cloning sites between these markers allowed the creation of a plastid targeting vector for the introduction of foreign genes (Staub, JM and Maliga, P., EMBO J. 12: 601-606 (1993), incorporated into the present as reference). Substantial increases in the frequency of transformation were obtained by replacing the recessive rDNA or the r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the deminifying enzyme of spectinomycin aminoglycoside-3 ' -adenyltransferase (Svab et al., 1993). Previously, this marker had been used successfully for the high-frequency transformation of the green alga plastid genome Chlamydomonas reinhardtii (Goldschmidt-Clemont, M., Nucí. Acids Res. 19: 4083-4089 (1991), incorporated herein by reference). Techniques for the transfection of plastids in plant protoplasts have also been described (O'Neill et al., Plant Journal 3 (5): 729-738 (1993) and Koop et al., Planta 199: 193-201 (1996), both incorporated herein by reference). An especially preferred plant plastid promoter for use in plastid targeting vectors for the purpose of expressing foreign genes in the plastid of the plant is the promoter of the clpP gene. The clpP gene encodes the proteolytic subunit of the ATP-dependent protease Clp, which in Arabidopsis is constitutively expressed in the plastids of the tissues of photosynthetic and non-photosynthetic plants (Shanklin et al., The Plant Cell 7: 1713-1722 (1995), incorporated herein by reference). clpP is also one of the few plant plastid genes that is retained in the genomes of non-photosynthetic plants (eg Epifagus virginiana; Morden et al., EMBO "10: 3281-3288 (1991)), and it is known that the clpP message is expressed in the plastids of the albostrians barley mutant, which lacks a detectable plastid translation activity (Hübschmann and Bórner, Plant Mol. Biol. 36: 493-496 (1998)) Therefore, it is possible for the clpP promoter to be transcriptionally active, even in non-green plastids.The characterization of the promoter region from the clpP gene of tobacco is described in International Publication Number WO 97/06250, incorporated herein by reference In this reference, the clpP gene of tobacco is characterized by having 5 'promoter sequences that are recognized by both a nuclear-encoded plastid RNA polymerase (nuclear encoded p_lastid , NEP by its acronym in English), and a plastid-encoded plastid RNA polymerase (p_lastid encoded plastid, PEP.) A primary transcript that appears from the sequence The clpP promoter of tobacco that maps to the nucleotide -53 position (upstream from the ATG translation start codon) is characterized in International Publication Number WO 97/06250 as highly expressed in the bleached plastids of tobacco mutants which lack an RNA polymerase encoded by the plastid, by virtue of the deletion of the rpoB gene. The clpP promoter sequence of tobacco has been used to drive the expression of a herbicide-resistant form of the Arabidopsis Protoporphyrinogen IX ("PROTOX") gene in tobacco plastids (International Publication Number WO 97/32011, incorporated herein by reference). reference). Identical constructs have been introduced that replace a GUS reporter gene in tobacco plastids, demonstrating that expression driven by clpP is not restricted to green plastids, but is also found in root plastids (leucoplastos, amiloplastos) and in the plastids of flowers (chromoplasts). Despite the promise shown by the transformation of plastids, only recently has this technology been applied to plants other than tobacco. International Application Number WO 97/32977, incorporated herein by reference, discloses methods and compositions for creating transplastomic plants in the Crucif rae family, such as Brassica and Arabidopsis, using leaf and cotyledon cells. However, what is also needed are novel plastid promoter sequences from different tobacco plants, particularly Arabidopsis, which can be used to boost the expression of transgenes in the green and non-green plastids of Arabidopsis, and in any other species of plant. In view of the foregoing, an object of the invention is to provide a novel plastid promoter from Arabidopsis thaliana, which is functional in all types of plastids. Another objective of the invention is to provide a method for using protein coding regions of plastid genes, in order to isolate novel intervening regulatory sequences, such as novel promoter sequences or untranslated 3 'or 5' RNA sequences. Still another object of the invention is to use the novel plastid promoter sequences to improve the plastid transformation efficiency, by reducing the unwanted homologous recombination between the native DNA sequences in the plastid genome and the exogenous DNA sequences contained in the chimeric DNA fragments incorporated in the plastid transformation vectors. Following these and other objects, the present invention provides a nucleic acid promoter isolated from the 5 'flanking region upstream of the coding sequence of the clpP gene of the Arabidopsis plastid. In a preferred embodiment, the nucleic acid promoter of the invention is substantially similar to a promoter sequence downstream of nucleotide number 263 of SEQ ID NO: 1. In a more preferred mode, the nucleic acid promoter of the invention has sequence identity with a promoter sequence downstream of nucleotide number 263 of SEQ ID NO: 1. In yet another embodiment, the promoter of the nucleic acid of the invention is substantially similar to SEQ ID NO: 1. In yet another embodiment, the promoter of the nucleic acid of the invention is comprised within SEQ ID N0: 1. In still another embodiment, the promoter of the nucleic acid of the invention comprises a nucleotide portion 20 base pairs and a sequence identical to a consecutive 20 base pair nucleotide portion of SEQ ID NO: 1. The present invention also encompasses a chimeric gene comprising the nucleic acid promoter of the invention operably linked to the coding sequence of a gene of interest; a plant transformation vector comprising this chimeric gene; and a plant, plant cell, plant seed, plant tissue, or plastid of transgenic plant, each comprising this chimeric gene. In another aspect, the present invention provides a novel method for isolating regulatory DNA sequences intervening between the protein coding regions of two plastid genes, which comprises the steps of: (a) determining the relative orientation and sequence of degenerate or specific nucleotides of protein coding regions of two plastid genes; (b) designing a first degenerate or specific polymerase chain reaction primer based on the determined sequence of the protein coding region of one of the two plastid genes; (c) designing a second degenerate or specific polymerase chain reaction primer based on the determined sequence of the protein coding region of the other of the two plastid genes; (d) amplifying a DNA fragment using the primer of steps (b) and (c), wherein the amplified DNA fragment comprises a regulatory DNA sequence intervening between the protein coding regions of the two plastid genes .

In a preferred embodiment of this method, the two plastid genes are a clpP gene and a psbB gene. According to this embodiment, the intervening regulatory DNA sequence comprises a clpP promoter. In another preferred embodiment of this method, the two plastid genes are a 16S rRNA gene and a valine tRNA gene. According to this embodiment, the intervening regulatory DNA sequence comprises a 16S rRNA promoter. In still another aspect, the present invention provides an improved plastid transformation method, which comprises transforming a plastid from a host plant species with a chimeric gene comprising an active regulatory sequence in the plastid operably linked to a coding sequence. of interest, wherein the regulatory sequence has a nucleotide sequence that is less than about 90 percent identical to a corresponding native regulatory sequence in the plastid of the host plant, where unwanted somatic recombination between the sequence is reduced regulator in the chimeric gene and the corresponding native regulatory sequence in the plastid of the host plant. In a preferred embodiment of this method, the chimeric gene is isolated from the plastid genome of the host plant species, and at least about 10 percent of the nucleotides in the regulatory sequence have been mutated. In another preferred embodiment of this method, the regulatory sequence in the chimeric gene is isolated from the plastid genome of a different species of plant than the species of the host plant. For example, the regulatory sequence in the chimeric gene can be isolated from the Arabidopsis plastid genome. In a particularly preferred embodiment, the regulatory sequence in the chimeric gene is a nucleic acid promoter isolated from the 5 'flanking region upstream of the coding sequence of the clpP gene of Arabidopsis. In another especially preferred embodiment, the regulatory sequence in the chimeric gene is a nucleic acid promoter isolated from the 5 'flanking region upstream of the coding sequence of the Arabidopsis 16S rRNA gene. Other objects and advantages of the present invention will become more apparent to those skilled in the art from a study of the following description of the invention and of the non-limiting examples.

DESCRIPTION OF SEQUENCES IN THE LIST OF SEQUENCES SEQ ID NO: l is the nucleotide sequence of the promoter region of the clpP gene of Arabidopsis. SEQ ID NO: 2 is the primer A_clpP used in Example 1. SEQ ID NO: 3 is the primer A_psbB used in Example 1. SEQ ID NO: 4 is the primer Aclp_Pla used in Example 2. SEQ ID NO : 5 is the Aclp_P2b primer used in Example 2. SEQ ID NO: 6 is the rpsl6P_ primer used in the Example 2. SEQ ID NO: 7 is the primer rpsl6P_lb used in Example 2. SEQ ID NO: 8 is the initiator of the upper chain used in Example 3.

SEQ ID NO: 9 is an initiator of the lower chain used in Example 3. SEQ ID NO: 10 is the nucleotide sequence of the promoter region of the 16S rRNA gene of Arabidopsis. SEQ ID NO: 11 is an initiator of the upper chain used in Example 4. SEQ ID NO: 12 is a lower chain initiator used in Example 4.

DEFINITIONS For clarity, certain terms used in the specification are defined and presented as follows: Associated With / Operably Linked: refers to two nucleic acid sequences that are physically or functionally related. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence encoding an RNA or a protein, if the two sequences are operably linked, or located in such a way that the regulatory DNA sequence will affect the level of expression of the coding or structural DNA sequence. Chimeric Gene / Fusion Sequence: a recombinant nucleic acid sequence wherein a promoter or regulatory nucleic acid sequence is operably linked to, or is associated with, a nucleic acid sequence that encodes an mRNA, or that is expressed as a protein, such that the regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequence of the chimeric gene is not normally operably linked to the associated nucleic acid sequence as found in nature. Coding Sequence: nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA, or anti-sense RNA. Preferably, the RNA is then translated into an organism to produce a protein. Gene: a defined region that is located within a genome and that, in addition to the aforementioned coding sequence, comprises other primary regulatory sequences responsible for the control of the expression, ie, transcription and translation, of the coding portion. A gene can also comprise other untranslated sequences and 5 'and 3' termination sequences. Other elements that may be present are, for example, introns. Gene of interest: any gene that, when transferred to a plant, confers on the plant a desired characteristic, such as resistance to antibiotics, resistance to viruses, resistance to insects, resistance to diseases, or resistance to other pests, tolerance to herbicides , better nutritional value, better performance in an industrial process, or altered reproductive capacity. The "gene of interest" can also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant. Heterologous Nucleic Acid Sequence: a nucleic acid sequence unnaturally associated with the host genome into which it is introduced, including multiple copies that do not occur naturally of a naturally occurring nucleic acid sequence. Homologous Nucleic Acid Sequence: a nucleic acid sequence naturally associated with a host genome into which it is introduced. Homologous recombination: the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules. Isolated: in the context of the present invention, an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or an enzyme that, by the hand of man, exists apart from its native environment, and therefore, does not It is a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form, or may exist in a non-native environment, such as, for example, a transgenic host cell. Minimum Promoter: promoter elements that are inactive, or that have greatly reduced their promoter activity in the absence of upstream activation. In the presence of an appropriate transcription factor, the minimal promoter functions to allow transcription. Nucleic Acid / Sequence, Acid Molecule Nucleic: a linear segment of DNA or RNA of a single chain or double chain that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a DNA segment. Plant: any plant at any stage of development, particularly a seed plant. Plant Cell: a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell can be in the form of a single isolated cell or a cultured cell, or as a part of a higher organized unit, such as, for example, a plant tissue, a plant organ, or a whole plant . Cell Plant Cultivation: plant unit cultures, such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different stages of development. Plant Material: leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. Plant Organ: a distinct and visibly structured and differentiated part of a plant, such as a root, "stem, leaf, flower bud, or embryo." Plant Tissue: as used herein, means a group of plant organized into a structural and functional unit, including any plant tissue in the plant or in cultivation.This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, and Any groups of plant cells organized into structural and / or functional units The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as mentioned above, or as otherwise encompassed by this definition, does not pretend to be exclusive of any other type of plant tissue Promotora: a sequence of untranslated DNA upstream of the coding region that contains the binding site for the RNA polim erasa II, and initiates the transcription of DNA. The promoter region may also include other elements that act as regulators of gene expression. Protoplast: an isolated plant cell without a cell wall, or with only parts of the cell wall. Regulatory Sequence: a non-translated nucleic acid sequence that assists in, enhances, or otherwise affects the transcription, translation, or expression of an associated structural nucleic acid sequence that codes for a protein or other genetic product. Regulatory sequences include promoters. A promoter sequence is typically located at the 51st end of a translated sequence, typically between 20 and 100 nucleotides from the 5 'end of the translation start site. Regulatory sequences may also include transcribed but untranslated nucleic acid sequences located 5 'and 3' of the coding sequences. These untranslated RNAs are normally involved in the post-transcriptional regulation of gene expression. Substantially Similar: with respect to nucleic acids, a nucleic acid molecule having at least 60 percent sequence identity with a reference nucleic acid molecule. In a preferred embodiment, a substantially similar DNA sequence is at least 80 percent identical to a reference DNA sequence; in a more preferred embodiment, a substantially similar DNA sequence is at least 90 percent identical to a reference DNA sequence; and in a more preferred embodiment, a substantially similar DNA sequence is at least 95 percent identical to a reference DNA sequence. A substantially similar nucleotide sequence is usually hybridized to a reference nucleic acid molecule, or fragments thereof, under the following conditions: hybridization in 7 percent sodium dodecyl sulfate (SDS), 0.5 M NaP04, pH 7.0 , 1 mM EDTA at 50 ° C; wash with 2X SSC, 1 percent SDS, at 50 ° C. With respect to proteins or peptides, a substantially similar amino acid sequence is an amino acid sequence that is at least 90 percent identical to the amino acid sequence of a reference protein or peptide, and has substantially the same activity as the reference protein or peptide. Tolerance: the ability to continue growth or normal function when exposed to an inhibitor or erbicide. Transformation: a process to introduce heterologous DNA into a cell, tissue, or plant, including a plant plastid. It is understood that cells, tissues, or transformed plants encompass not only the final product of UIL transformation process, but also their transgenic progeny. Transformed / Transgenic / Recombinant: refers to a host organism, such as a bacterium or a plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host genome, or the nucleic acid molecule can also be present as an extrachromosomal molecule. This extrachromosomal molecule can be self-replicating. It is understood that cells, tissues, or transformed plants encompass not only the final product of a transformation process, but also their transgenic progeny. An "untransformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, eg, a bacterium or a plant, that does not contain the heterologous nucleic acid molecule. Nucleotides are indicated by their bases by the following conventional abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). The amino acids are indicated in the same way by the following conventional abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (lie; L), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In addition, (Xaa; X) represents any amino acid. The present invention provides the promoter region for the clpP gene from the plamid Arabidopsis thaliana genome which encodes a plant homologue of the ATP-dependent protease Clp. The promoter disclosed can be used to drive the expression of coding sequences for selectable marker genes or any other genes of interest in the plastids of the transgenic plants. The promoter of the present invention is useful for the constitutive expression of transgenes in both green and non-green plastids, and therefore, is particularly useful for the transformation of the plastid in plants such as corn, where the selection of regenerable transformants requires the selection in non-green weaves. A clpP promoter of Arabidopsis of the present invention can be incorporated into plastid transformation vectors, and can be transformed into plastids according to methods known in the art, particularly those described in the following: United States of America Patents Numbers 5,451,513; 5,545,817; 5,545,818; and 5,576,198; International Applications Nos. WO 95/16783, WO 97/32011, and WO 97/32977; and Svab et al. (1993) and McBride et al. (1994). The present invention also provides a novel method for using regions encoding plastid gene protein to isolate novel intervening regulatory sequences, such as novel promoters or 3 'or 5' UTRs. This method is exemplified by the Applicant's technique for isolating the clpP promoter region from the Arabidopsis plastid, and the promoter region of the 16S rRNA from the Arabidopsis plastid, as stipulated in detail in the examples below. Briefly stated, the isolation of these promoter regions is facilitated by the opportunity to preserve the genetic order in the Arabidopsis plastid genome in relation to that of Nicotiana tabacu, for which the entire genome sequence of the plastid In tobacco, clpP is present in a divergent orientation of the psbB gene, whose coding sequence is conserved among a number of plant species. Because only 445 base pairs separate the start codon of psbB from the divergently oriented start codon of clpP in tobacco, the sequences of protein coding regions of the divergent clpP and psbB genes are used to design primers for the chain reaction of the polymerase that amplifies the non-coding intergenic region between these genes. This region includes the promoters for psbB in one orientation, and clpP in the other. An expressed sequence marking (EST) sequence of Arabidopsis is found in an EST database that appears to include a portion of the clpP coding sequence and the 5 'non-translated RNA (5'UTR). The sequence of this EST is used to design primers for amplification with the polymerase chain reaction of the clpP promoter, based on the Arabidopsis DNA sequence encoding the putative initiation of the clpP protein. These primers were paired with ones designed to match the highly conserved DNA sequences around the psbB start codon. Using these primers, a DNA fragment of about 500 nucleotides, which includes the clpP promoter region of the Arabidopsis plastid, is amplified from the total Arabidopsis DNA. A DNA fragment that includes the 16S rRNA promoter region of the Arabidopsis plastid is amplified in a similar manner.

Using the above method, one of ordinary skill in the art can use the protein coding regions of two nearby plastid genes to isolate intervening untranslated sequences, such as promoter sequences and other regulatory sequences, from the plastid genome. Preferably, the two plastid genes are adjacent, because there are no other sequences transcribed between the two nearby plastid genes, however, it is foreseeable that this method works even when there is a small gene, such as a gene that encodes a tRNA, in the region amplified between the two nearby plastid genes In a preferred embodiment, one of ordinary skill in the art can use the above method to isolate a plastid clpP promoter from the plastid genome of any plant. preferred embodiment, an ordinary expert in the subject can use the above method to isolate a prom otor of 16S rRNA of the plastid, from the plastid genome of any plant. The present invention further provides a method for using novel plastid promoters, such as the clpP or 16S rRNA promoters of the Arabidopsis plastid, to improve the plastid transformation efficiency, by "reducing the undesired recombination between the DNA sequences". native in the plastid genome, and the exogenous DNA sequences contained in the chimeric DNA fragments that are incorporated into the plastid-transformation vectors.It is known that even the relatively short regions of homology between the native DNA sequences in the Plastid genome and exogenous DNA sequences will eventually cause somatic recombination in plastid transformants.This biological property has even been used as a means to eliminate selectable markers of plastid transformants in the Chlamydomonas green algae chloroplasts, by the flanking of the marker is reactable with identical repeated heterologous DNA sequences. Although the minimum required homology size stretch or the precise degree of sequence identity within a particular homology stretch sufficient for recombination has not been identified, as few as 50 base pairs of homology to the plastid genome may be sufficient to induce recombination. These recombination events are visible in transgenic plants as pale sectors in the leaves, which result from the division of the cells in which the plastid genome has been reconfigured In extreme cases, the result is almost white leaves with small green patches, indicating that recombination occurs in most somatic cells and their lineage.The essential characteristics of non-recombinogenic regulatory sequences (such as promoters and 5 'and 3' UTRs) include both the ability to function Correspondingly, for the purpose of controlling the expression of the heterologous gene in the plastids of a plant species of interest, such as the lack of sufficient sequence identity to promote the recombination of the plastid homologue, the latter property can be achieved either by using a heterologous regulatory sequence derived from the plastid genome of a different plant species, which have a divergent sequence to less than 85 to 90 percent identity, or by sufficiently mutating a native regulatory sequence derived from the plastid genome of the same plant species. In one embodiment, this method involves using the clpP promoter of Arabidopsis of the present invention to direct the transcription of genes of interest in the plastids of heterologous plant species such as tobacco, corn, rice, soybean, tomato, potato, or others In another embodiment, this method involves using the Arabidopsis 16S rRNA promoter described in the examples, to direct the transcription of genes of interest in the plastids of heterologous plant species such as tobacco, corn, rice, soybeans, tomato, potato, etc. In addition to plastid genes from higher plants, heterologous promoters useful or the 5 'and 3' UTRs for the non-recombinogenic regulation of plastid transgenes, can also be derived from plastid genes of lower plants or algae, chromosomal genes of cyanobacteria, or genomes of viruses that infect chloroplasts of the plant or the algae, or the cyanobacterial cells.The selection of native genes mutated from the same plant, which are unable to have an unwanted recombination, is facilitated by the random mutagenesis of regulatory sequences, in such a way that it is reduced sequence identity up to more than 90 percent relative to the initial sequence, then the cluster of random regulatory sequences is selected. mutated for the subset that is still active in the plastid (capable of having normal functioning in plant plastids) by cloning each mutant upstream of a selectable marker gene that operates in the plastid, and then transforms everything the group of chimeric DNAs in the plastids of wild-type plants. Only those mutated sequences still capable of functioning in plastids will result in the expression of the selectable marker in the transgenic plants. The transgenic plants expressing the selectable marker are also evaluated to determine somatic recombination, by observing the frequency of sectoring of the leaf. The target region of the plastid genome of a transformed plant which expresses the selectable marker, and which has a desirable frequency of sectoring the leaf, is then sequenced to determine which mutated regulatory sequence is present. Accordingly, this mutated sequence satisfies the criteria for controlling the expression in a plastid of a gene of interest, and having a sufficient sequence divergence in relation to the DNA sequences of the native plastid to reduce the frequency of undesired recombination.

EXAMPLES The invention will be further described with reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting, unless otherwise specified. The "molecular" conventional recombinant DNA and cloning techniques used herein are well known in the art, and are described by Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Labora tory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), and by TJ Silhavy, ML Berman, and LW Enquist, Experiments wi th Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984).

Example 1: Isolation of the ClpP Promoter Region of Arafoidps ± s. The isolation of the clpP promoter region of Arabidopsis is facilitated by the opportunity to preserve the genetic order in the Arabidopsis plastid genome in relation to that of Nicotiana tabacum, a plant for which the entire plastid genome sequence is known. . In tobacco, clpP is present in a divergent orientation of the psbB gene, which has been sequenced from a number of plant species, and is shown to be conserved in its sequence. An alignment of the psbB sequences of tobacco, corn, wheat, and Nicotiana acumina, indicates that the first eight amino acids are conserved identically, as well as their DNA coding sequences. In tobacco, only 445 base pairs separate the start codon psbB from the divergently oriented start codon of clpP. In view of the foregoing, the Applicant postulates that the sequences of the protein coding regions of the divergent clpP and psbB genes can be used to design primers for the polymerase chain reaction, which could amplify the non-coding intergenic region between these genes. This region, in theory, will include promoters for psbB in one orientation, and clpP in the other. An expressed sequence marker (EST) sequence from Arabidopsis is found in the TIGR NHC AtEST database (http://-www.tigr.org) which appears to include a portion of the clpP coding sequence and RNA not translated 5 '(5'UTR). Because the putative translation of this sequence is similar to the mature clpP of E. coli, and therefore does not appear to include a plastid transit peptide, it is postulated that this EST (Seq ID # P_3982 from the base of TIGR data NHC AtEST) represents a portion of the plastid clpP message. However, because Shanklin et al. (1995) have suggested that there may be homologs of nuclear encoding clpP in Arabidopsis, the Applicant is cautious to find these in place of the genuine gene encoded by the plastid. Because ESTs by definition come from expressed messages, EST_3982 is not expected to include any non-transcribed clpP promoter regions. The nucleotide sequence of EST_3982 is used to design primers for clpP promoter polymerase chain reaction amplification, based on the Arabidopsis DNA sequence encoding the initiation of the clpP protein in this plant. These primers are put in pairs with ones designed to match the highly conserved DNA sequences around the psbB start codon, which the Applicant postulates that are similarly conserved in Arabidopsis. The primers used are: A_clpP: 5 '-. AAGGGACTTTTGGAACGCCA? TAGGCAT-3 * (SEQ ID NO: 2) and A- psbB: 5 '-CACGATACCAAGGCAAACCCATGGA-3' (SEQ ID NO: 3).

They successfully amplify a DNA fragment of approximately 500 nucleotides from the total DNA of A. thaliana. ("Landsburg erecta" variety) using Pfu thermostable DNA polymerase. The blunt-ended DNA fragment was sequenced both directly (using the cloning primers) and subsequent to cloning into the EcoRV site of the vector pGEM5Zf (-) to construct the plasmid pPH146b. The nucleotide sequence of this polymerase chain reaction fragment of approximately 500 base pairs, is given in SEQ ID N0: 1. Sequence analysis reveals 86 percent sequence identity with the clpP promoter region of tobacco over a region of 200 base pairs extending upstream of the clpP start codon. Accordingly, SEQ ID NO: l includes the clpP promoter region of Arabi dopsi s.

Example 2: Preparation of a Chimeric Gene Containing the Prompting Sequence of Arabidopsis ClpP and the 5 'Untranslated Sequence of Native ClpP Fused with a GUS Reporter Gene and the 3' Untranslated Sequence of the Tobacco Plastid rpslβ Gene in a Vector of Transformation of Plastids. I. Amplification of the ClpP Gene Promoter from the Plastid Arabidopsis and RNA Not Translated 5 'Complete (5'UTR). The plasmid DNA pPH146b is used as the template for the polymerase chain reaction, with a "top chain" primer from left to right comprising an EcoRI restriction site introduced at position -234 in relation to the codon ATG start sequence of the clpP gene of the Arabidopsis plastid (nucleotide number 263 of SEQ ID N0: 1) (Aclp_Pla primer: 51-GCGGAATTCATCATTCAGAAGCCCGTTCGT-3 '(SEQ ID NO: 4, underlined ECORI restriction site)), and an initiator of the "bottom chain" from right to left homologue for the region from -21 to -1 in relation to the ATG start codon of the clpP promoter that incorporates a BspHI restriction site introduced at the start of translation (Aclp_P2b primer: 5 '-GCGTCATGAAATGAAAGAAAAAGAGAAT-3' (SEQ ID NO: 5, BspHI restriction site underlined)). This polymerase chain reaction is undertaken with Pfu thermostable DNA polymerase (Stratagene, La Jolla, CA) on a Perkin Elmer 480 Thermal Cycler according to the manufacturer's recommendations (Perkin Elmer / Roche, Branchburg, NJ) as follows: 7 minutes at 95 ° C, followed by 4 cycles of 1 minute at 95 ° C / 2 minutes at 43 ° C / 1 minute at 72 ° C, then 25 cycles of 1 minute at 95 ° C / 2 minutes at 55 ° C / l minute at 72 ° C. A product of the 250 base pair amplification, comprising the promoter and the 5 'untranslated region of the clpP gene of Arabidopsis, containing an EcoRI site at its left end, and a BspHI site at its right end, with two modifications near the ATG to correspond with the 5'UTR of the tobacco clpP sequence, it is gel purified using conventional procedures, and digested with EcoRI and BspHI (all restriction enzymes can be purchased from New England Biolabs, Beverly, MA ).

II. Amplification of the Untranslated RNA Sequence 3 * of the rpslß Gene of Tobacco Plastid (3'UTR). Total DNA from N. tabacum variety "Xanthi NC" is used as the template for the polymerase chain reaction as described above, with a "top chain" primer from left to right comprising a site of Xbal restriction introduced immediately followed by the TAA stop codon of the plastid rsplβ gene encoding the S16 ribosomal protein (rspl6P_la primer: 5'-GCGTCTAGATCAACCGAAATTCAATTAAGG-3 '(SEQ ID NO: 6, Xbal underlined restriction site)), and a initiator of the "lower chain" from right to left homologue for the region from +134 to +151 in relation to the stop codon TAA of rps! 6 that incorporates a HindIII restriction site introduced at the 3 'end of the 3' Rsplβ UTR (rspl6P_lb primer: 5'-CGCAAGCTTCAATGGAAGCAATGATAA-3 '(SEQ ID NO: 7, underlined HindIII restriction site)). The product of the amplification comprising the 3 'untranslated region of the rpslß gene, which contains an Xbal site at its left end, and a HindIII site at its right end, and which contains the region corresponding to nucleotides 4943 a 5093 of the DNA sequence of the N. tabacum plastid (Schinozaki et al., 1986), is gel purified and digested with Xbal and HindIII.

III. Linkage of a GUS Reporter Gene Fragment with the clpP Gene Promoter and the 5 'and 3' UTRs. A reporter gene fragment of β-glucuronidase (GUS) of 1864 base pairs, derived from plasmid pRAJ275 (Clontech), containing a Ncol restriction site at the ATG start codon, and an Xbal site following the 3 'Native UTR, is produced by digestion with Ncol and Xbal. This fragment is ligated in a four-way reaction with the 250 bp Arabidopsis EcoRI / BspHI clpP promoter fragment, the 3 'UTR fragment of rpsl G of tobacco Xbal / HindIII of 157 base pairs, and an EcoRI / HindIII fragment of 3148 base pairs from the cloning vector pGEM3Zf (-) (Promega, Madison Wl), to construct the plasmid pPH165. The plastid transformation vector pPH166 is constructed by digesting plasmid pPRVllla (Zoubenko et al., 1994) with EcoRI and HindIII, and ligating the resultant 7287 base pair fragment with an EcoRI / HindIII fragment of 2222 base pairs of pPH165.

Example 3: Isolation of the Promoting Region of the Arabidopsis 16S rRNA Gene. Isolation of the promoter region of the rRNA gene 16S of Arabidopsis is facilitated by the opportunity that the genetic order in the genome of the Arabidopsis plastid is conserved in relation to that of Nicotiana tabacum, a plant for which the entire plastid genome is known. In Sinapis alba, a species closely related to Arabidopsis, the 16S rRNA gene and the valine tRNA are oriented as in tobacco (GenBank, accession number CHSARRN1). The promoter region of the Arabidopsis 16S rRNA gene is isolated by polymerase chain reaction amplification (Pfu Turbo DNA polymerase, Stratagene, La Jolla, CA) using A. thaliana total (variety "Landsburg erecta") as a template, and the following primers that are conserved in both Nicotiana and Sinapis alba: initiator of the "upper chain" (5 '-CAGTTCGAGCCTGATTATCC-3' (SEQ ID NO: 8)), and the primer of the "lower chain" (5) '-GTTCTTACGCGTTACTCACC-3' (SEQ ID NO: 9)). The predicted amplification product of 379 base pairs (based on the Sinapis alba sequence) comprising the promoter region of the Arabidopsis 16S rRNA gene, which corresponds to nucleotides 102508 to 102872 of the tobacco plastid genome (Shinozaki et al., 1986) is ligated with blunt ends at the EcoRV site of pGEM5Zf (-) (Promega), to construct pArab 16S, and sequence analysis and comparisons are made with the tobacco 16S rRNA promoter. The product of the promoter region of the Arabidopsis 16S rRNA gene is 369 base pairs, and is stipulated as SEQ ID NO: 10. Example 4: Preparation of a Chimeric Gene Containing the Promoter of Arabidopsis 16S rRNA Gene and Untranslated Sequence 51 Native Fused with the Ribosome Linkage Site of the Tobacco rbcL Gene, a GUS Reporter Gene, and the 3"Untranslated Sequence of the Tobacco Plaster rpslß Gene, in a Plastid Transformation Vector I. Amplification of the 16S rRNA Gene Promoter of the Arabidopsis Plastid and the 5 'Native Untranslated Sequence (5' UTR), and Fusion with the Ribosome Linkage Site of the Tobacco rbcL Gene.The pArabldS plasmid DNA is used as the template for the polymerase chain reaction, with an "upper chain" primer comprising an EcoRI restriction site introduced at the 5 'end of the promoter region of the 16S rRNA gene (position 63 of SEQ ID NO: 10) (5'-GCCGGAATTCTCGCTGTGATCGAATAAGAATG-3' ( SEQ ID NO: 11, restriction site EcoRI underlined)). The "lower chain" primer extends to position 172 (SEQ ID NO: 10) of the untranslated region 51 of the 16S rRNA gene promoter, mutates three ATGs downstream of the transcription initiation site by changing position 151 (T to G) (SEQ ID NO: 10), position 158 (A to C) (SEQ ID NO: 10), and position 167 (A to C) (SEQ ID NO: 10), merge with the ribosome binding site of the rbcL gene of tobacco (positions 57569 to 57585) (Shinozaki et al., 1986) as an extension 51 for the 31 end of the 5 'UTR of the 16S rRNA gene, and introduces a BspHI site at the end 3 'of the ribosome binding site (5' -GCCTTCATGAATCCCTCCCTACAACTATCCAGGCGCTTCAGATTCGCCTGGAGTT-3 '(SEQ ID NO: 12, BspHI underlined restriction site)). The amplification with polymerase chain reaction is carried out with the Pfu Turbo DNA Polymerase cassette (Stratagene). The product of the 145 base pair amplification, comprising the promoter of the Arabidopsis 16S rRNA gene, and the 5 'untranslated region with three mutated ATGs, and the ribosome binding site of the rbcL gene of tobacco, is purified in gel and digested with EcoRI and BspHI, producing a product of 131 base pairs.

II. Linkage of the Arabidopsis 16S rRNA Gene Promoter, 5 'UTR, and Ribosome Binding Site of the rbcL Tobacco Gene, with the Reporter Gene GUS and the Untranslated Region 31 (3' UTR) of the rpslß Gene of Tobacco Plasmid , in a Vector of Plastid Transformation. A reporter gene fragment of -glucuronidase (GUS) of 1864 base pairs derived from plasmid pRAJ275 (Clontech), which contains a Ncol restriction site at the ATG start codon, and an Xbal site following the stop codon, is produced by digestion with Ncol and Xbal. This fragment is ligated in a four-way reaction with the promoter of the Arabidopsis EcoRl / BspHI 16S rRNA gene of 131 base pairs, 5 'UTR, and the fragment of the ribosome binding site rbcL of tobacco, the fragment of 3 'UTR of rpsl ß of tobacco Xbal / HindIII described ~ in Example 2, and an EcoRI / HindIII fragment of 3148 base pairs from the cloning vector pGEM3Zf (-) (Promega, Madison, Wl). A plastid transformation vector is constructed by digesting the above construct with EcoRI and HindIII, and ligating the resulting 2.1 kb fragment with an EcoRl / HindlII fragment of 7.3 kb from the pPRVllla plasmid (Zoubenko et al., 1994).

Example 5: Biolistic Transformation of the Tobacco Plastid Genome. . Seeds of Nicotiana tabacum variety 'Xanthi nc' are germinated, in seven per dish, in a circular arrangement of 2.54 centimeters on a medium of T agar, and are bombed 12 to 14 days after sowing with tungsten particles of 1 miera (MIO, Biorad, Hercules, CA) coated with DNA of the plasmids described above in Example 2 and Example 4, essentially as described in Svab, Z. and Maliga, P. ((1993 PNAS 90, 913-917 The bombarded seedlings are incubated in a medium T for two days, after which the leaves are cut and placed with the abaxial side up in bright light (350-500 micromoles-photons / square meter / second) on plates of RMOP medium (Svab, Z. Hajdukiewicz, P. and Maliga, P 81990) PNAS 87, 8526-8530), containing 500 micrograms / milliliter of spectinomycin dihydrochloride (Sigma, St. Louis, MO). The resistant shoots that appear under bleached leaves three to eight weeks after the bombardment, are subcloned in the same selective medium, are allowed to form callus, and the secondary shoots are isolated and subcloned. The complete segregation of copies of the transformed plastid genome (homoplasmicity) in independent subclones is evaluated by conventional Southern blot techniques (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor). The total digested cellular DNA is separated with BamHI / EcoRI (Mettler, IJ (1987) Plant Mol Biol Repoter 5, 346-349) on 1 percent Tris-borate agarose gels (TBE), transferred to nylon membranes (Amersham), and probed with 32P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI / HindIII DNA fragment from pC8 which contains a portion of the address sequence to the plastid rps7 / 12. The homoplasmic shoots are aseptically rooted in an MS / IBA medium containing spectinomycin (McBride, K. E. et al. (1994) PNAS 91, 7301-7305), and transferred to the greenhouse. Those skilled in the art will be able to see various modifications of the invention described herein. It is intended that these modifications fall within the scope of the appended claims.

LIST OF SEQUENCES < 110 > Hovartis AG < 120 > NOVEDOSA PROMOTORA SEQUENCE D? PLASTIDOS DE PLANTAS < 130 > PH / 5-30422 / CGC 1988 < 140 > < 141 > < 160 > 12 < 170 > Patentln Ver. 2.0 < 210 > 1 < 211 > 499 < 212 > DNA < 213 > Arabidopsis thaliana < 220 > < 221 > various_characteristics < 222 > (497) .. (499) < 223 > ATG start codon of clpP < 220 > < 221 > various_ characteristics < 222 > Complement ((6) .. (8)) < 223 > ATG start codon of psbB < 400 > 1 aaacccatgg aaatacccct ttatcaacga aaaatagaca ctatgtaact ttattgcatt 60 ggaaaaaact atgctacgta cccccccttt ttaggtaatt atttcggaga aaggattaat 120 atttgttcta ttctgttagt aataatggaa caattcaatt catagaaaaa aagggaagcg 180 atatccgata gatctattct tgcaatgggg agtaccaata ttttctatga gttaatccta 240 accaagatag ctattgttgt tgatcattca gaagcccgtt cgtaaaaaat ttcctttagt 300 tttattcatt ctctcttact ttacttttat tttatatttt attttagctt attcaactta 360 tgtattaaat atcattaatt taaatatgat taataaagta ggaaaaaagg ataatagtat 420 taaaaaacga aaccccaatt ttacgtttcc acatcaaagt gaaatagaga acttcattct 480 cttttttttt catttcatg 499 < 210 > 2 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: PCR primer A_clpP derived from ESTP_3982 < 400 > 2 aagggacttt tggaacgcca ataggcat 28 < 210 > 3 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: PCR primer A_j? SbB derived from the DNA sequences conserved around the start codon psbB < 400 > 3 cacgatacca aggcaaaccc atgga 25 < 210 > 4 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Initiator Aclp_ > the < 400 > 4 gcggaattca tcattcagaa gcccgttcgt 30 < 210 > 5 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Initiator Aclp_P2b < 400 > 5 gcgtcatgaa atgaaagaaa aagagaat 28 < 210 > 6 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: initiator rpsl6P_la < 400 > 6 gcgtctagat caaccgaaat tcaattaagg 30 < 210 > 7 < 211 > 27 < 12 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: initiator rpsl6_lb < 400 > 7 cgcaagcttc aatggaagca atgataa 27 < 210 > 8 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: initiator of the upper chain < 400 > 8 cagttcgagc ctgattatcc 20 < 210 > 9 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: initiator of the lower chain < 400 > 9 gttcttacgc gttactcacc 20 < 210 > 10 < 211 > 369 < 212 > DNA < 213 > Arabidopsis thaliana < 400 > 10 cagttcgagc ctgattatcc ctaaacccaa tgaatgtgag tttttctatt ttgacttgct 60 ccctcgctgt gatcgaataa gaatggataa gaggctcgtg ggattgacgt gagggggtag 120 atttctggga gggtagctat gcgaactcca tgcgaatatg aagcgcatgg atacaagtta 180 tgaaagacaa tgacttggaa ttccgaatca gctttgtcta cgaagaagga agctataagt 240 aatgcaacta tgaatctcat ggagagttcg atcctggctc aggatgaacg ctggcggcat 300 gcttaacaca tgcaagtcgg acgggaagtg gtgtttccag tggcggacgg gtgagtaacg cgtaagaac 360 369 < 210 > 11 < 211 > 32 < 12 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: initiator of the upper chain < 400 > 11 gccggaattc tcgctgtgat cgaataagaa tg 32 < 210 > 12 < 211 > 55 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: initiator of the lower chain < 400 > 12 gccttcatga atccctccct acaactatcc aggcgcttca gattcgcctg gagtt 55

Claims (20)

1. A nucleic acid molecule comprising a nucleic acid promoter isolated from the 5 'flanking region upstream of the coding sequence of a clpP gene from the Arabidopsis plastid.

2. A nucleic acid molecule according to claim 1, wherein said nucleic acid promoter is substantially similar to a promoter sequence downstream of nucleotide number 263 of SEQ ID NO: 1.

3. A nucleic acid molecule according to claim 1, wherein said nucleic acid promoter has sequence identity with a promoter sequence downstream of the number of nucleotide number 263 of SEQ ID NO: 1.

4. A nucleic acid molecule according to claim 1, wherein said nucleic acid promoter is substantially similar to SEQ ID NO: 1.

5. A nucleic acid molecule according to claim 1, wherein the nucleic acid promoter is comprised within SEQ ID N0: 1.

6. A nucleic acid molecule according to claim 1, wherein the nucleic acid promoter comprises a nucleotide portion of 20 base pairs identical in sequence to a consecutive 20 base pair nucleotide portion of SEQ ID N0: 1

7. A chimeric gene comprising the nucleic acid molecule of claim 1 operably linked to the coding sequence of a gene of interest.

8. A plant transformation vector comprising the chimeric gene of claim 7.

9. A plant, plant cell, plant seed, plant tissue, or transgenic plant plastid, each comprising the chimeric gene of the claim 7. A method for isolating ras-regulated DNA sequences intervening between the protein coding regions of two plastid genes, which comprises the steps of: (a) determining the relative orientation and a degenerate nucleotide sequence or specific for protein coding regions of two plastid genes; (b) designing a first degenerate or specific polymerase chain reaction primer based on the determined sequence of the protein coding region of one of the two plastid genes; (c) designing a second degenerate or specific polymerase chain reaction primer based on the determined sequence of the peptide coding region of the other of the two plastid genes; (d) amplifying a DNA fragment using the primers of steps (b) and (c), wherein the amplified DNA fragment comprises a regulatory DNA sequence intervening between the protein coding regions of the two plastid genes . 11. A method according to claim 10, wherein the two plastid genes are a clpP gene and a psbB gene. 12. A method according to claim 11, wherein the intervening regulatory DNA sequence comprises a clpP promoter. 13. A method according to claim 10, wherein the two plastid genes are a 16S rRNA gene and a valine tRNA gene. 14. A method according to claim 13, wherein the intervening regulatory DNA sequence comprises a 16S rRNA promoter. 15. An improved plastid transformation method, which comprises transforming a plastid from a host plant species, with a chimeric gene comprising an active regulatory sequence in plastids operatively linked to a coding sequence of interest, wherein the regulatory sequence has a nucleotide sequence that is less than about 90 percent identical to a corresponding native regulatory sequence in the plastid of the host plant, where unwanted somatic recombination is reduced between the regulatory sequence in the chimeric gene, and the native regulatory sequence corresponding to the plastid of the host plant. 16. A method according to claim 15, wherein the regulatory sequence in the chimeric gene is isolated from the plastid genome of the species of the host plant, and wherein at least about 10 percent of the nucleotides of this regulatory sequence have mutated. 17. A method according to claim 15, wherein the regulatory sequence in the chimeric gene is isolated from the plastid genome of a different plant species than the species of the host plant. 18. A method according to claim 17, wherein the regulatory sequence in the chimeric gene is isolated from the genome of the Arabidopsis plastid. 19. A method according to claim 18, wherein the regulatory sequence in the chimeric gene is a nucleic acid promoter isolated from the 5 'flanking region upstream of the coding sequence of a clpP gene of the Arabidopsis plastid. 20. A method according to claim 18, wherein the regulatory sequence in the chimeric gene is a nucleic acid promoter isolated from the 5 'flanking region upstream of the coding sequence of a plastid 16S rRNA gene. of Arabidopsis.