WO2007033587A1

WO2007033587A1 - A method for isolating a bi-directional gene promotor and the use thereof

Info

Publication number: WO2007033587A1
Application number: PCT/CN2006/002448
Authority: WO
Inventors: Youliang Peng; Wensheng Zhao; Junhua Liu
Original assignee: China Agricultural University
Priority date: 2005-09-19
Filing date: 2006-09-19
Publication date: 2007-03-29
Also published as: CN1936002A

Abstract

The present invention discloses bi-directional gene promotor, its variant or functional equivalent and the use thereof. Also provided is a recombinant nucleic acid sequence, a recombinant construct, an expression system and a plant cell comprising the promoter, variant or functional equivalent thereof. In addition, the present invention further discloses a method for isolating the bi-directional gene promotor and for preparing a transgenic plant.

Description

Method for separating bidirectional promoter and application thereof

The invention belongs to the field of plant genetic engineering. In particular, it relates to an isolated bidirectional gene promoter, a recombinant nucleic acid sequence comprising the promoter, a construct and an expression system, and the use thereof to drive expression of a functional gene in a plant. It also relates to a method of isolating a two-way gene promoter.

Background technique

Plants are affected by a large number of different types of microorganisms during their growth. Therefore, the diseases caused by these microorganisms have always been one of the main causes of crop failure. However, in the long-term interaction with these microorganisms, plants have also evolved a variety of defense mechanisms against the invasion of surrounding pathogenic microorganisms, so that they can survive and multiply in competition with various microorganisms. In general, plant resistance to pathogens can be divided into two categories: constitutive passive defense and induced active defense, in which induced active defense plays a leading role in plant resistance to pathogens. The so-called active defense refers to a series of defense responses that plants can quickly identify and activate when they are infected by pathogens. Hypersensitive response (HR) is a typical type of active defense response, which is characterized by: Host plants often rapidly develop local cell death around the pathogen infection site. For living nutrients and some non-living nutrient pathogens, this local cell death of the plant can effectively limit the spread of the pathogen into the surrounding cells, which is manifested as disease resistance.

In many plant disease systems, the gene-to-gene relationship between the host cultivar (or strain) and the pathogen race (or pathogenic type), that is, the resistance of the cultivar is the resistance gene (R gene) contained therein. The result of interaction with the corresponding avirulence gene (^vr gene) in the pathogen. In this interaction, signal recognition, transduction, and cascade amplification are involved, and a series of defensive responses are initiated to render the plant resistant to disease. This disease resistance is called gene-to-gene resistance, or race-specific resistance. However, when the pathogen population does not contain a corresponding avirulence gene or a non-toxic gene, the host plant can not effectively identify and change the pathogen that changes or mutates, and initiates a defense response, which ultimately manifests as a disease. . The cultivation and utilization of disease-resistant varieties has long been the main effective means of controlling plant diseases. However, long-term large-scale cultivation of a single resistance gene will cause high selection pressure on the pathogen population, resulting in the emergence of new dominant toxic races, which will cause plants to "lost" disease resistance. At present, resistant varieties are mainly obtained through sexual hybridization, usually through hybridization, backcrossing and a series of offspring screening processes, with a long cycle, and sometimes the breeding rate of resistant varieties can not keep up with the emergence of new dominant species. Speed (Ben J. Corneslissen, Plant Physiol., 1993, 101:700-712) _o This, sexual hybridization is restricted by interspecific reproductive isolation, distant plants Genes of useful genes or other organisms cannot be introduced as intended, and therefore, the source of resistance is limited.

In recent years, with the cloning of some useful resistance genes and defense genes, it is expected to develop resistant varieties through transgenic pathways. If a disease-resistant gene of a clone is transferred to another variety or other species, the breeding process can be shortened by eliminating complicated steps such as hybridization, backcrossing and offspring selection, but the resistance of the transgenic plant is still a small species specialization. Resistant, resistant to only one or some specific races and not resistant to other races, therefore, also facing the loss of disease resistance due to the emergence of new races (Ben J. Comeslissen, Plant Physiol. , 1993, 101 : 700-712). Using a constitutive promoter (such as the commonly used CaMV 35S promoter) to drive certain genes (such as defense genes) to enhance the resistance of transgenic plants to transgenic plants is considered to be one of the effective strategies for breeding relatively long-lasting resistant varieties, but A strategy has its own flaws that are difficult to overcome. First, as mentioned above, the allergic reaction of plants is caused by the death of their own local cells to kill or limit the expansion of pathogens to express resistance. For plants, the genes expressed in this "sacrificial partial preservation" strategy are often inductively expressed. If these genes (such as the HR gene) are expressed constitutively, they are often destructive to plants (De Wit, 1992, Annu. Rev. Phytopathol., 30: 391-418). Second, strong constitutive promoter-driven transgene expression tends to cause post-transcriptional gene silencing, and the higher the transcriptional level of the transgene, the greater the probability of gene silencing (Elmayan T and Vaucheret H, 1996, The Plant J., 9 ( 6): 787-797). Therefore, the ideal strategy for cultivating broad-spectrum or long-lasting disease-resistant crops through transgenic pathways is to use pathogen-inducible inducible gene promoters to drive non-specific inducible expression of key genes responsible for disease resistance, allowing plants to be infected only by pathogens. Express defense response, thereby limiting the growth of pathogens and even killing them (Peng, 1994, Integrated Resource Management for Sustainable Agriculture, pp450-453, which can alleviate the degree of damage to plants during disease resistance, and avoid or reduce transgenics Silence is happening. To this end, many laboratories are looking for pathogen-inducible inducible promoters. This laboratory cloned a rice blast fungus non-small-specific infection-inducible gene promoter MPP774 from rice. The highest activity of induced expression was 3 to 10 times that of CaMV35S (Zhang Shihong, Ph.D., China Agricultural University, 2002), and the promoter was used to drive different defense genes to transform rice, in order to obtain rice strains with broad-spectrum disease resistance. Despite this, there is a lack of available pathogen-inducible efficient promoters. However, the main factor limiting the implementation of this strategy.

On the other hand, many times when cultivating plant varieties through genetic engineering, multiple different genes need to be transferred to the same plant. However, the gene promoters that are currently widely used are mostly unidirectionally driven and can only drive one gene expression. Therefore, the introduction of multiple genes requires the construction of multiple vectors with one or more different promoters, which leads to cumbersome and inefficient transgenic processes. Moreover, studies have shown that transgenic plants driven by the same promoter are susceptible to transcriptional transgene silencing (de Eilde C, et al, 2000, Plant Mol. Biol. 43: 347-359), and this gene is silenced with the coding sequence of the transgene (Vau _C heret H, et al, CR Acad. Sci. Paris, 1993, 317: 310-323) and the length of the promoter (De Neve, M Et al, Mol. Gen. Genet., 1999, 260, 582-592.) Irrelevant. Even different promoters with sequence homology can cause gene silencing at the transcriptional level. Vaucheret H et al. found that a 90 bp homologous sequence between promoters is sufficient to cause gene silencing at the transcriptional level (Vaucheret H, et al, CR Acad. Sci. Paris, 1993, 317: 310-323). Therefore, when transferring different genes into the same plant, the same promoter or a promoter with homologous sequences should be avoided as much as possible. From this point of view, the use of a gene promoter having a bidirectional driving gene expression ability should be effective in avoiding the occurrence of the above-mentioned transcription level gene silencing. To this end, Xie MT et al. proposed a strategy for artificially constructing a bidirectional gene promoter to drive expression of two genes, and proved that: a unidirectional promoter is fused in the opposite direction to a basic promoter region (TATA box region), which has a bidirectional drive. The ability to express genes (Xie MT, et al, 2001, Nature Biotechnol. 19: 677-679).

From the perspective of cultivating broad-spectrum disease-resistant crops, it is necessary to isolate and identify plant gene promoters that are non-small-specifically induced by pathogens and have two-way driving gene expression ability, for genetic engineering to cultivate long-lasting or broad-spectrum disease-resistant crops. Very urgent. Moreover, the determination and release of whole genome sequences of plants such as rice and Arabidopsis contribute to the isolation of this bidirectional gene promoter. Summary of the invention

Summary of invention

In view of the above problems, the inventors have established long-term exploration and extensive research to establish a cloned cDNA by negative subtractive screening-reverse Northern confirmation, and then isolate candidate bidirectional promoter regions according to bioinformatics analysis, and then prove by transgenic pathway. The method of bidirectional promoter function in this area. Finally, a bidirectional gene promoter with bidirectional driving gene expression was successfully obtained, and its sequence and function were deeply studied. It was found that the promoter can drive endogenous or exogenous genes in transgenic monocots such as rice leaves. Inducible expression of pathogens, constitutive expression in the roots, and also driving of constitutive expression of endogenous or exogenous genes in the roots and leaves of transgenic dicots such as Arabidopsis thaliana. It is believed that this promoter will have important application value in broad-spectrum disease resistance genetic engineering. The present invention has thus been completed. Accordingly, the object of the present invention is firstly to provide a bidirectional gene promoter having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or a variant sequence thereof, which is directed against SEQ ID O: a sequence obtained by deleting, adding, replacing or modifying the bases 162 to 932 in the sequence shown, or the bases from position 217 to position 987 in the sequence shown in SEQ ID NO: 2. A sequence obtained by deletion, addition, substitution or modification. Specifically, the bidirectional gene promoter sequence is set forth in SEQ ID NO: K 2, 3, 4, 14 or 15. It is also an object of the present invention to provide a functional equivalent of the bidirectional gene promoter having the SEQ ID

NO: The sequence shown in any of 7-13, and the TATA box is fused at the 5' end.

The present invention also provides a promoter having the sequence shown in any one of SEQ ID NOS: 7-13.

The present invention also provides an enhancer characterized in that the activity of the promoter is enhanced after fusion to the promoter 3, which has positions 49 to 176 in SEQ ID NO: 5 and SEQ ID NO: Any of the sequences shown in bits 45 through 497.

The present invention also provides a key cis element having the sequence shown in any one of SEQ ID NOS: 16, 17.

In another aspect of the invention, a recombinant nucleic acid sequence comprising the above promoter is provided. Preferably, the recombinant nucleic acid sequence comprises at least one functional endogenous or exogenous gene.

In the present invention, the term "functional endogenous or exogenous gene" may be a disease resistance gene, a defense gene and its related genes isolated from rice or other plants, or a gene of a key enzyme or protein in a defense process, Or the phytoalexin synthase gene having direct or indirect bactericidal action, or may be an antisense RNA isolated from rice blast fungus or a gene closely related to rice blast fungus, or may be isolated from animals or microorganisms. Genes of disease-resistant active substances or genes for synthesizing their enzymes, and beneficial genes such as drought-resistant, cold-resistant, salt-resistant or high-temperature resistance genes.

In another aspect of the invention, a recombinant construct comprising the recombinant nucleic acid sequence described above is provided.

In another aspect of the invention, an expression system comprising the recombinant construct described above is provided.

In still another aspect of the invention, there is provided the use of a promoter as described above, a recombinant nucleic acid sequence comprising the promoter, a construct or an expression system for driving expression of an endogenous or exogenous gene in a plant.

In the present invention, the plant is a monocot or a dicot. The monocot is preferably rice, and the dicot is preferably Arabidopsis.

The present invention also provides a method for isolating a bidirectional gene promoter comprising cDNAs of a plurality of genes isolated and expressed from an organism, such as a plant, and analyzing the cDNA on the chromosome by bioinformatics means known in the art. Specific position; then, to find out two cDNAs located on the opposite side of a certain DNA (named DNA1) and having opposite transcription directions; by transgenic analysis and confirming whether DNA1 is a bidirectional gene promoter, that is, the DNA1 sequence As a post-selective two-way driver, it is analyzed by transgenic means to confirm the organ and tissue specificity of its expression and its response to abiotic stresses such as drought, cold, salinity, high temperature and other abiotic stresses and pests. Finally, it is determined by gradual deletion. The cis-elements, and select the appropriate cis-elements and sequences, and construct artificial promoters of corresponding length and expression intensity by DNA recombination. This strategy is especially applicable Other promoters having a two-way driving gene expression function or cloning from other plants having a bidirectional driving gene expression function are isolated from organisms whose whole genome sequence has been determined, such as rice, Arabidopsis, and the like. Such promoters drive the expression of genes linked to their sides to have different tissue or organ specificities, either inductively expressed, specifically expressed in tissues, organs or developmental stages, or constitutively expressed.

Specifically, the method of the present invention comprises: (1) preparing a rice cDNA library infected by a pathogenic bacteria such as Magnaporthe oryza, and plating the membrane; (2) hybridizing the mRNA of healthy rice leaves as a probe, and selecting a clone that is not hybridized. As a candidate inducible cDNA clone and confirmed by reverse Northern; (3) Compare the confirmed inducible cDNA sequence with the published rice genome database, look for head-to-head (ie, 5' end relative) and a piece of DNA The two strands of the complementary strand of the fragment (designated DNA1) are matched; (4) The function of the bidirectional gene promoter of DNA1 is verified by linking DNA1 to an exogenous transcribable gene and detecting the expression of the exogenous transcribable gene.

Specifically, the present invention uses rice as a material to clone a promoter having the ability to drive two genes of interest in both directions. The promoter can induce the expression of two genes of interest in the rice leaves infected by Magnaporthe oryzae (Fig. 8, 14), and can also express two target genes in the rice roots specifically (Fig. 15). The promoter also constitutively expresses two genes of interest in the roots and leaves of transgenic Arabidopsis thaliana (Figures 16, 17, 18). More specifically, the sequence of the bidirectional promoter is SEQ ID NO: 1 and SEQ ID NO: 2, and the above two sequences are reverse complementary sequences, that is, sequences of two complementary single strands of the same DNA fragment, for convenience of description, They are named pSCI2 and pSCI3, respectively. Wherein, the TATA box and the transcription start site (TSS) of pSCI2 are located at 1095 bp and 1125 bp of SEQ ID NO: 1, respectively, and the TATA box and TSS of pSCI3 are located at 1096 bp and 1026 bp of SEQ ID NO: 2, respectively; The transcribed DNA sequence (including endogenous and exogenous DNA sequences) can be placed at or ligated to the 3' end of pSCI3 to construct a binary expression vector, which can be transgenic after introduction into rice or Arabidopsis through appropriate routes. pSCI2 or/and pSCB in rice and Arabidopsis can drive transcription of this transcribable DNA sequence.

The present invention also provides introns belonging to two different protease/chymotrypsin inhibitors (0^«'2 and (3⁄43⁄4^) genes of rice at the 3' end of pSCI2 and pSCI3, respectively, which are respectively contained in SEQ ID NO: 5 and SEQ ID NO: 6. The intron sequence of the OsSci2 gene from 49 bp to 176 bp in SEQ ID NO: 5 is from 45 bp to 497 bp in SEQ ID NO: 6. An intron sequence of d3. SEQ ID NO: 5 is placed at the 3' end of SEQ ID NO: 1 and fused thereto, and SEQ ID NO: 6 is placed at the 3' end of SEQ ID NO: 2 and fused thereto, respectively The resulting two-stage DNA sequence: ^C/2+/ and ^C/3+J, which have the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, are characterized by: pSCI2+ I has stronger activity in inducing expression of the target gene than pSCI2 (Fig. 11); ^C 3+/ is more potent in inducing expression of the target gene (Fig. 12). The intron can also be combined with other promoters. Fusion, and have Enhancement of target gene expression.

In another aspect, the invention also provides several variant variants obtained by base deletion of pSCI2 and pSCI3. The derived variants pSCI2-l, pSCI2-2, pSCI2-3 and pSCI2-4 were obtained by PCR amplification from the 5'-end deletion of pSCU, respectively. The derived variant pSCI3-l was obtained from the 5'-end deletion of pSCI3. , pSCI3-2 and pSCI3-3. The PCR amplification primers are shown in Figure 7. pSCU-1, pSCI2-2, pSCI2-3 and pSCI2-4 have the nucleotide sequences from positions 69, 226, 403 and 616 to position 1152 in the sequence of SEQ ID NO: 1, respectively, the sequences of which are respectively SEQ ID NO: 7, 8, 9, 10; 7SCZW, and ? have the nucleotide sequences from positions 93, 214 and 400 to position 1152 in the sequence of SEQ ID NO: 2, respectively, the sequences of which are respectively SEQ ID NO : 11, 12, 13 are shown. It is characterized in that: these derived variants have different degrees of promoter activity, and any of the derived variants can be selected to drive different degrees of expression of the target gene according to the needs of use (Fig. 9 and Fig. 10). It will be understood by one of ordinary skill in the art that the DNA sequence fused to the TATA box at the 5' end of SEQ ID NO: 7-13 also functions as a bidirectional gene promoter of the present invention.

The invention also provides two variant variants pSCISP2 and pSCISP3 obtained by restriction endonuclease digestion of pSCI2 and pSCI3. pSCISP2 is a self-ligated fragment of the pSCU by restriction endonuclease and has the nucleotide sequence of SEQ ID NO: 14 and is characterized by the deletion of nucleotides 162 to 932 of SEQ ID NO: 1. _; after 7SC / SP3 Spei is digested with endonuclease restriction endonucleases and self-ligated resulting from the pSCI3 having SEQ ID NO: 15 is the nucleotide sequence, wherein deletion of SEQ ID NO: 2 in the 217 to 987 Nucleotide. One of ordinary skill in the art will appreciate that the variant variants are also obtainable by PCR. They all have the ability to bidirectionally drive the constitutive expression of the gene of interest in rice leaves, and pathogen infection enhances its expression activity (Fig. 13).

One of ordinary skill in the art will appreciate that the promoter of the above-described rice two-way gene promoter and its derivative variants is ligated to an endogenous or exogenous transmissible gene, and the promoter or its derivative is determined by detecting the expression of the gene. Variant promoter activity. For example, the promoter activity of the above-described rice bidirectional gene promoter and its derived variant can be confirmed by the expression of Gt/S by fitting the reporter gene Gt/S downstream of the promoter. A preferred embodiment is the sophisticated method established by Jerfferson et al. (Jerfferson, EMBO J., 1987, 6:3901~3907).

One of ordinary skill in the art will appreciate that based on the above teachings of the present invention and the common general knowledge in the art, the above-described i^C/2, pSCI3, pSCI2+I, and various derivative variants thereof are deleted, base-altered, A DNA fragment obtained by base addition or re-isolation of a part, a part or a combination thereof and having the ability to drive expression of a gene of interest is within the scope of protection of the present invention. Using any of the above rice bidirectional gene promoters, derived variants thereof, enhancers and key cis elements

The expression vector constructed by the DNA sequence or the binary expression vector and the cell line obtained by the transformation, the recombinant microorganism, and the transgenic plant are all covered by the patent. That is, the vector constructed by the above-described promoter-active DNA sequence fragment or by using the optional DNA sequence fragment is used to construct a vector for transforming a plant and its cell line or microorganism. The recombinant DNA technology involved in the present invention can be implemented by one of ordinary skill in the art.

The bidirectional promoter and its derived variants provided by the present invention can be ligated to any DNA fragment of interest to construct a related vector, expression vector or binary expression vector, and the DNA fragment function can be determined in rice or other plants. Any DNA fragment is a reporter gene or a Luciferase gene in the present invention, but a gene to be expressed which is preferred in practical use may be a gene that directly inhibits, interferes with or destroys the survival of a pathogen, a disease resistance gene, a defense gene, a signaling gene, and a gene. A heterologous gene (such as a non-toxic gene, antisense RNA, etc.) which is expressed in plants such as rice and which can effectively control diseases, and may also be a gene having other uses.

The bidirectional promoter provided by the invention can be used for the cultivation of disease resistant rice. The bidirectional promoter provided by the present invention is ligated to two genes which play a key role in disease resistance and constructs a binary expression vector, introduced into Agrobacterium tumefaciens, and transformed into rice by Agrobacterium tumefaciens to obtain disease resistance. Or disease-resistant genetically modified rice. A preferred embodiment of the disease resistant rice of the present invention is to cultivate rice resistant to rice blast, and another preferred embodiment is rice resistant to root diseases. Further, the application of the bidirectional promoter provided by the present invention is not limited to rice, but can be applied to other monocotyledons or dicotyledons.

Thus, in another aspect, the invention provides a plant cell comprising a promoter and a functional endogenous or exogenous gene as described above in the invention.

In another aspect, the invention provides a method of obtaining a transgenic plant comprising transforming an expression system of the invention as described above into a plant.

According to the characteristics of the bidirectional promoter and the derivative variant thereof of the present invention, the reverse sequence of the same gene is ligated at both ends thereof to construct an RNA interference vector and transferred into a plant, and the function and utilization of the related gene can be carried out.

The "cloning" of the DNA fragment in the present invention means a promoter sequence which is isolated from the natural rice genome in nature and can be directly used alone without any change. It will be understood by those skilled in the art that, according to the sequence of the present invention, a pair of upstream and downstream primers can be designed, which can be easily obtained again by conventional PCR techniques, or can be screened from a rice genomic library by using a labeled probe, and even a classic can be used. Synthetic or chemical synthesis is obtained, which can be done by one of ordinary skill in the art.

The present invention also provides a key cis-acting element that functions in the pSCI2 and pSCI3 promoters, characterized by having the sequences set forth in SEQ ID NO: 16 and SEQ ID NO: 17. In the present invention, these cis-acting effects Deletion of the elements results in a significant decrease in expression of the reporter gene driven by the pSCI3 promoter, ie, they have an enhanced effect on the expression of the reporter gene driven by the promoter. These cis-acting elements can also be linked to other promoters to enhance expression of the gene of interest.

The method of introducing a disease-resistant binary expression vector into rice in the present invention is simple in the art and can be accomplished by those skilled in the art. The detection of the activity of the above-mentioned promoter or its derivative variants and the use of the promoter for targeted disease-resistant molecular manipulation are inevitably carried out in plant systems, a preferred embodiment being Agrobacterium-mediated Methods (Hid et al, Plant 1.1994, 6:271-282) introduced it into rice; users can also flexibly choose the following transformation methods according to their proficiency: protoplast fusion method, gene gun bombardment method, electric excitation method, PEG method, leaf disc method, etc. (Horsch et al, Science, l 984, 234: 496; Barton et al, Cell, 1983, 32: 1033; Liu et al, Acta P ytophysiol. Sin, 21: 195-205; Horsch et al. Science, 277: 1229)

Other derivative variants in which the promoter according to the invention is active, after transformation of the transgenic gene, during tissue culture from rice resistant callus to rooting and moving into the natural environment (such as the field), the transformed tissue or individual has similar constitutive expression. Activity, which is caused by the difference in the tissue culture medium during the stimulation of the active ingredient or tissue culture during the tissue culture (such as similar damage), which is basically lost after the transplanted into the field.

The molecular manipulation tool enzymes designed in the present invention are TaKaRa products unless otherwise specified. BRIEF DESCRIPTION OF THE DRAWINGS:

The invention is further illustrated by the following figures.

Figure 1. Screening by negative subtraction - Reverse Northern confirmation of cloned rice blast fungus-inducible cDNA. Caption: In the circular part of the above figure, the rice cDNA library was randomly plated and transfected, and the mRNA of healthy rice leaves was used as a probe to hybridize; the clones that were not hybridized (indicated by open circles in the above figure) were selected as candidate induction. Sex cDNA clone. The following figure shows that the candidate inducible cDNA clones were arrayed on two nylon membranes, respectively, and the reversed Northern confirmation was carried out by hybridizing the mRNA of healthy rice leaves and inoculated rice blasts, respectively. The left side of the figure was inoculated with rice blast fungus leaves. The mRNA (R) probe, right, is the healthy leaf mRNA (H) probe. Among them, the hybridization signal (black dot in the figure) was determined to be an inducible cDNA clone.

Figure 2. Reverse Northern real confirmation of rice blast-induced rice cDNA clones. Caption: After the candidate cDNA was amplified by PCR, the same array was arrayed on two nylon membranes, and hybridized with rice healthy leaf mRNA (left) and rice blast fungus inoculated rice leaf mRNA (right) as probes. Most positive clones have a hybridization signal only on the right side of the membrane, which is an inducible clone.

Figure 3. The top panel shows the rice subtilisin/trypsin inhibitor gene ay& and staining The structure of the body. Caption: The structure is indicated above, the thick solid arrow in the right direction indicates the cDNA transcribed by the Oy&i2 gene and its orientation, the left solid arrow indicates the cDNA transcribed from the ⁄3⁄3 3 gene and its orientation; the open box indicates the intron; ATG For translation initiation codon; TSS is the transcription start site. Black lines indicate DNA. The two genes are located on the complementary sequence of the DNA; the lower panel shows the Southern hybridization map of the rice variety Aizhixu. The left and right probes are the coding regions of OsSci2 and OsSci3, respectively. Lanes M, 1, and 2 are DNA molecular weight standards, Hind III digestion genome and EcdK digestion genome.

Figure 4. Northern analysis of the dynamic expression of OsSci2 and OsSci3 genes after infection by Magnaporthe oryzae. Illustration: 0, 8, 24, 48, 72 are the inoculation time, the unit is hour (h), the inoculation of rice blast fungus is non-affinity race 131 and affinity race 007, ribosomal RNA is within the sample volume Standard.

Figure 5 is a schematic representation of the construction of a two-way vector of the rice blast-induced rice bidirectional 3⁄4 promoter region and its derived variants and reporter gene conjugation. Caption: The above figure is a schematic diagram of the fusion of the promoter and its derivative variants with the reporter gene GUS. The solid line indicates the promoter region, the solid arrow of the pentagon indicates the GUS reporter gene, the same below; the following figure shows the removal of CaMV35S Promoter pCAMBIA1301 vector, the restriction enzyme site marked on the vector £c. And Ncol is the junction site of the promoter fragment. Cc MV 35S is the 35S promoter of cauliflower mosaic virus.

Figure 6. Schematic diagram of the binary expression vector PCAMBIA1301 of the rice blast fungus-inducible rice bidirectional gene promoter and the gene of interest (gene I and gene Π).

Figure 7. Genetically modified mutants of the rice blast-induced rice bidirectional gene promoter region and their degenerate deletions from the 5' end. Caption: The primer pair name corresponds to the promoter or its variant name, underlined as an increased restriction site, the upstream primer increases the site by EcoRI, and the downstream primer increases No I.

Figure 8. Comparison of the activity of the reporter gene found in the transgenic rice leaves inoculated with Magnaporthe oryzae and the reporter gene driven by the O^^S promoter. Schematic illustration: Left is a schematic representation of pSCI2 and / and Gf/ gene linkage, the number indicates a transcription start site of +1. On the right is the GUS expression activity, Inoculation is the sample 36 hours after the inoculation of Magnaporthe oryzae, Mock is the sample 36 hours after the water treatment, and Untreated is the sample without any treatment. The activity of the reporter gene Gt/S driven by pSCI2 and pSCI3 was 28 and 12-fold, respectively, of the Ca^S-driven reporter gene Gi/S expression.

Figure 9. Comparison of the activity of pSCI2 and its derivative variant driving gene expression in transgenic rice leaves inoculated with Magnaporthe oryzae. Schematic description: Left is a schematic diagram of the linkage between the 3⁄4SC/2 and its derived variants and the Gt« gene. The number in the figure indicates that the transcription start site is +1, which is an empty vector without a promoter; the right is the corresponding GUS expression. Activity, Inoculation is a sample 36 hours after inoculation of Magnaporthe oryzae, and Mock is a sample 36 hours after water treatment. Untreated is a sample that has not been treated at all.

Figure 10. Comparison of the activity of ^C? and its derivative variants driving gene expression in leaves of transgenic rice inoculated with Magnaporthe oryzae. Schematic illustration: Left is a schematic diagram of the linkage between the derivative variant and the Gf/S gene. The figure indicates that the transcription start site is +1, ^ is an empty vector without a promoter; the right is the corresponding GUS expression activity. Inoculation was a sample 36 hours after inoculation with Magnaporthe oryzae, Mock was a sample 36 hours after water treatment, and Untreated was a sample without any treatment.

Figure 11. Comparison of the intensity of Gt/S gene expression in transgenic rice leaves after inoculation with Magnaporthe oryzae. Schematic description: Left is and; schematic diagram linked to G?7S gene, the figure indicates that the transcription start site is +1, WP is an empty vector without promoter; right is corresponding to GUS expression activity, Inoculation is rice For the 36 hours after the inoculum treatment, Mock was the sample 36 hours after the water treatment, and Untreated was the sample without any treatment.

Figure 12. Comparison of the intensity of ASCZ? and driven Gt/S gene expression in transgenic rice leaves after inoculation with Magnaporthe oryzae. Caption: The left is a schematic diagram of the connection between SCZ? and ;C/3+/ and the Gf/S gene. The numbers in the figure indicate that the transcription start site is +1, and WP is an empty vector without a promoter; Corresponding to GUS expression activity, Inoculation was a sample 36 hours after inoculation of Magnaporthe oryzae, Mock was a sample 36 hours after water treatment, and Untreated was a sample without any treatment.

Figure U. Analysis of GUS expression activity of pSCISP2 and pSCISP3-driven Gt/S gene-expressing transgenic rice leaves after inoculation with Magnaporthe oryzae. Caption: The left is a schematic representation of the GUS gene expression vector driven by pSClSJP2 and pSCISP3. The figure indicates that the transcription start site is +1, which is an empty vector without a promoter; the right is the corresponding GUS expression activity, and the Inoculation is rice. The sample was 36 hours after the inoculum treatment, and Mock was a sample 36 hours after the water treatment, and Untreated was a sample without any treatment.

Figure 14. Tissue staining of the two-way promoter pSC/2, and its derived variant-driven Gf/S gene, induced by Magnaporthe oryzae in the leaves of transgenic rice. Legend: 1 to 16 are: pSCI2, _P SCI2-l, pSCI2-2, pSCI2-3, pSCI2-4, pSCI2SP, pSCU+I, WP, pSCI3, pSCI3-l, pSCJ3-2, pSCIS-3 , CaMV35S, pSCI3SP, SC/J+Z and / 3⁄4SC 2 (not vaccinated). The arrow in the figure shows one of the stained (blue) sites, and the uninoculated and inoculated T T plants have no blue color, indicating that the bidirectional promoter and its derived variants are inducible expression of Magnaporthe oryzae in rice leaves.

Figure 15. Two-way promoter; Tissue staining of the expression of the SC/pSCB region-driven Gi/S gene in the roots of transgenic rice. Schematic description: Gt/S staining of transgenic rice with no promoter vector, the main root apex is listed on the left side, the lateral root germination part is listed in the middle, and the lateral root is listed on the right side. pSCI2, pSCB transgenic water The rice plants had GUS expression at the root tip, lateral root germination point and lateral root (indicated by the arrow in the figure, blue). The WP transgenic plants showed no coloration at the above sites, indicating that the two-way promoter was constitutively expressed in rice roots. .

Figure l6. GUS expression activity analysis of transgenic Arabidopsis roots expressed by pSCI2 and pSCI3 driven Gi/S gene. Schematic description: Left is a schematic diagram of the driven G3⁄4S gene expression vector. The figure indicates that the transcription start site is +1, which is an empty vector without a promoter; the right is the corresponding GUS expression activity.

Figure Y GUS expression activity analysis of transgenic Arabidopsis leaves expressed by pSCU and pSCI3-driven Gt/S gene expression. Schematic description: Left is a schematic diagram of the gene expression vector driven by SC/3. The figure indicates that the transcription start site is +1, which is an empty vector without promoter; the right is the corresponding GUS expression activity.

Figure 18. Tissue staining of the two-way gene promoter pSC/2, pSC73 region-driven gene in transgenic Arabidopsis thaliana. Schematic description: From left to right, transgenic Arabidopsis thaliana T2 plants expressing no promoter (i P), CaMV 35, pSCD and pSCI2 driven GOT genes. The pSCI2, pSCB transgenic rice Arabidopsis thaliana had GUS expression in the leaves and roots (arrows in the figure, blue), and the transgenic plants showed no coloration, indicating that the bidirectional promoter was found in the roots and leaves of Arabidopsis thaliana. For constitutive expression. Specific implementation

The invention will be further elucidated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. In the following examples, experimental methods which do not specify the specific conditions are usually carried out in accordance with conventional experimental methods or in accordance with the manufacturer's recommended operating instructions.

Example 1. Cloning of Rice Infectious Rice Gene Induced by Magnaporthe oryzae

In order to clone a pathogen-inducible gene promoter capable of bidirectionally driving gene expression, the present invention firstly selects inducibility from a cDNA library of rice blast fungus infecting rice leaves by a negative subtractive screening-reverse Northern confirmation method. The cDNA clone was 1642. The specific implementation is as follows: planting rice variety Aizhixu Oryzc sativa japonica cv. Aichisahi, China National Genebank Uniform No.: WD-11165), when 4-5 leaves are taken out, part of the healthy rice leaves are taken, immediately after liquid nitrogen freezing and preservation , extracting total RNA from leaves and purifying mR A (denoted as HmRNA); another part of living organism inoculated with Magnaporthe oryzae P131 (Peng, YL and Shishiyama, J. CanJ. Bot. 1988, 66: 730-735), respectively The diseased leaves were taken at 0, 8, 24, 48, and 72 hours after inoculation. Immediately after storage, the liquid nitrogen was frozen and stored, and the total RA of the leaves was extracted. The appropriate amount of RNA was mixed and purified at each time point (recorded as PmRNA). At the same time, the cDNA library of the rice leaf infected by Magnaporthe oryzae was plated into a monoclonal plate and imprinted on a nylon membrane. The construction of the cDNA library was carried out according to the instructions of the λ ZAP expressTM kit provided by Stratagene. The sample used was inoculated with rice cultivar P131. Aichi Zhi 0, 4, 8, 12, 24, 36, N2006/002448

After 48 hours, the leaf mRNA was mixed in equal amounts. The H mRNA was subjected to radioisotope α- ³² Ρ labeling with Superscrip II reverse transcriptase (manufactured by TaKaRa Co., Ltd.), and the above nylon membrane was hybridized with α- ³² Ρ labeled cDNA as a probe. After hybridization, negative clones were picked for PCR amplification, and the amplified products were arrayed on two nylon membranes. Reverse Northern hybridization was performed with α- ³² Ρ-labeled H mRNA and PmR A cDNA probes, respectively (Fig. 1 and Fig. 2). Clones (1790) that hybridized only to the cDNA probe of P mRNA were positive as candidate clones and sequenced. Example 2. Bioinformatics analysis of inducible cDNA sequences

The 1790 cDNA sequences in Example 1 subjected to negative subtractive screening-reverse Northern confirmation were compared with the rice genome database (http://ricegaas.dna.affrc.go.jp), and it was found that 1642 of them were found. The sequence is derived from rice. Further, cDNAs derived from rice were compared with each other, BLAST query and genomic database analysis, and they were found to belong to 1075 independent rice genes, respectively. At the same time, it was found that the two genes were homologous to other reported biologically derived subtilisin/trypsin inhibitor genes, and were named OsSci2 and OsSci3 (GenBank accession numbers AY878694 and AY878695, respectively); and the two cDNAs are located in the same On a BAC clone, the two were matched in a head-to-head manner (ie, 5' end relative) to a complementary strand of a DNA fragment (designated DNA1), and the two cDNAs were 1152 bp apart. The chromosomal positions of the two genes are shown in Figure 3. . · Example 3. Specific and temporal dynamic analysis of OsSci2 and OsSci3 induced expression by rice blast fungus infection.

Northern blot analysis was used to analyze the expression characteristics and time dynamics of OsSci2 and OsSciS induced by Magnaporthe oryzae. The specific implementation method is as follows: Planting rice variety Aizhixu, when 4-5 leaves are extracted, part of the rice leaves are inoculated with the rice cultivar affinity 007 (Peng, YL and Shishiyama, Can. J. Bot. 1988, 66). : 730-735 ) , Another part of the living body was inoculated with the rice cultivar P131, and the leaves were taken at 0, 8, 24, 48, and 72 hours after inoculation. Immediately after storage, the liquid nitrogen was frozen and the total RNA was extracted. Parallel gel electrophoresis was performed as described in Molecular Cloning (Sambrook et al, 1998) with a sample loading of approximately 20 μ _§ per sample. The hybridization probes are the coding regions of the 0 _S Sci2 and OsSci3 labeled with the radioisotope α- ³²分别, respectively. After hybridization, the membrane was washed, pressed, and imaged in a CycloneTM phosphor screen scanning system (PeridnElmer). The results are shown in FIG. The results showed that the induced expression of C3⁄43⁄4 2 and OsSci3 had no race specificity, and the time dynamics of expression of the two genes were similar: all began to express at 8 hours after inoculation, reaching a peak at 48 hours. Example 4. Cloning of the bidirectional promoter pSCI2 and pSCI3 and its bidirectional promoter pSCI2+I and its intron

According to the results of bioinformatics analysis, in the rice genome, the two genes are located on the complementary strand of the same DNA fragment and distributed in a head-to-head manner. The two cDNAs are 1152 bp apart, and the 5' untranslated regions of both genes contain one. Intron. First, a specific primer pair pSCI2+I (SEQ ID NO-.18, 19) and pSCI3+I (SEQ ID NO: 25, 26) were designed based on the genomic sequence to amplify the genomic DNA of the rice variety Aizhixu. The PCR reaction conditions were: 94 4 minutes; 94 °C 30 seconds, 58 Ό 30 seconds, 72 2 minutes, 30 cycles; 72 10 minutes. The amplified fragment was cloned into a PMD-18T vector (manufactured by TaKaRa Co., Ltd.) and sequenced, and a nucleotide sequence of the DNA fragment shown in SEQ ID NO: 0.3 and SEQ ID N0.4 of the Sequence Listing was obtained. On this basis, primer pairs pSCI2 (SEQ ID NO: 18, 20) and pSCI3 (SEQ ID O: 25, 27) were designed and amplified using the above cloned fragment as a template. The PCR reaction conditions were: 94 ° C 4 Minutes; 94Ό 30 seconds, 55Ό 30 seconds, 72 V L5 minutes, 30 cycles; 72°C for 10 minutes. The amplified product was cloned into the pMD-18T vector and confirmed by sequencing to obtain a DNA sequence as shown in SEQ ID NO: 0.1 and SEQ ID NO. Example 5. Acquisition of a series of derived variants of the rice blast fungus inducible bidirectional gene promoter.

The above-mentioned cloned rice blast fungus-inducible bidirectional gene promoter; ^ CJ2 and ; ^ sequence database (PlantProm DB: http://mendel.cs.rhul.ac.uk or http:〃 www, softberry.com /) Search and software analysis (http: 〃 www.dna.affrc.go.ip/PLACE/ and http:〃 sphinx.mg.ac.be: 8080/ plant CARE/), speculating on the potential cis component position, PCR primers carrying appropriate restriction sites are then designed before these positions (Fig. 7, SEQ ID NO: 21, 20, SEQ ID NO: 22, 20, SEQ ID NO: 23, 20, SEQ ID NO: 24, 20, SEQ ID NO: 28, 27, SEQ ID NO: 29, 27, SEQ ID NO: 30 ^ 27), using the cloned original sequence (SEQ ID NO: 1) as a template for amplification (PCR reaction conditions are: 94 °C 4 minutes; 94°C 30 seconds, 55 30 seconds, 72°C 1.5 minutes, 30 cycles; 72ΧΠ0 minutes.) A series of derived variants of the 5' upstream regulatory fragment, cloned into PMD The -18T vector was confirmed by sequencing. Finally obtained; +I, pSCI2-l, pSCI2-2, pSCI2-3, pSCI2-4, pSCI3 + I, pSCB- pSCI3-2 SCI3-3 and pSCU and pSCI3 (see Figure 5). Example 6. Construction of a binary vector in which a rice blast fungus-inducible bidirectional gene promoter pSCI2 and pSCI3 and a series of derived variants thereof were fused to a GMS gene.

The above-mentioned rice blast fungus-inducible bidirectional gene promoter PSCI2 cloned into the pMD-18T vector pSCB and its series of derived variants contain an EcoRL site at its 5' terminus and an Ncol site at its 3' terminus (Fig. 5). Therefore, the binary vector pCAMBIA1301 (product of CAMBIA) and the derivative variant cloned into pMD-18T were digested with restriction enzymes EcoRI and Ncol, respectively. After the double-digested products were separated by Sephadex electrophoresis, the gel fragments of the target DNA fragments were excised and recovered by a recovery kit (produced by Beijing Saibaisheng Co., Ltd.): (1) The binary vector pCAMBIA1301 was deleted 35S The remainder of the promoter, (2) pSCI2 and pSCI3 and its derived variants were excised from the PMD-18T vector. Finally, the recovered (1) (2) parts were mixed and bound with T4 DNA ligase according to a certain ratio, and the ligated product was transformed into E. coli DH5a strain, and confirmed by enzyme digestion and sequencing. The promoters and derived variants thus obtained are: pSCI2, pSCI3, pSCI2+I, pSCI3+I, pSCI2-l, pSCI2-2, pSCI2-3, pSCI2-4, pSCI3-l, pSCI-2 and pSCI-3 (See Figures 8, 9, 10, 11 and 12 on the left). In order to obtain derivative variants, SC/SP2 and ^, respectively, based on ; SC/2 and ; C / 3, respectively, using restriction endonuclease digestion, recovery of large fragments, self-ligation (see Figure 13 left) . Example 7. Two-way gene promoters pSCI2 and pSCI3 and a series of derived variants thereof and gene expression binary vectors were transformed into rice.

1. A binary vector transformed into Agrobacterium tumefaciens

The above binary vector plasmid DNA was separately introduced into competent cells (competent cells) of Agrobacterium strain EHA105 (E. Hood, et al, 1993, Transgen. Res. 2: 208-218, available from CAMBIA) by electroporation. The preparation and electroporation were carried out according to the method of Molecular Cloning (Sambrook et al, 1998), and cultured at 28'C to form a single colony. The transformed Agrobacterium was inoculated into YM liquid medium containing 5 (^g/ml Kanamycin, 28 Ό, shaken at 220 rpm for 16 hr, and the above liquid was directly subjected to the above PCR identification.

2. Agrobacterium tumefaciens-mediated rice transformation

Agrobacterium tumefaciens-mediated transformation of rice is carried out according to the method of Hiei et al. (Hiei et al., Plant Mol. Biol., 1997, 35: 205-218) o Specifically: preparation of immature seeds of rice, surface-sterilized and extruded rice The immature embryos were placed on solid induction medium and dark cultured to induce callus. After about 5-7 days, the callus is peeled off, transferred to freshly prepared subculture medium, subcultured for about 5 days under the same conditions; or prepared for mature embryo callus of rice, and subculture is selected for 5-7 days. The yellowish calli are co-cultured. The callus with better state was selected and co-cultured with a suitable amount of transformed Agrobacterium suspension (OD 0.3-0.5) for 15-20 minutes, transferred to solid artificial medium, and cultured in the dark at 26 ° C for 2-3 days. The co-cultured callus was placed on a differentiation medium containing 50 mg/L Hygromycin (manufactured by Roche), and then cultured for 3 days in a dark state, and then transferred to a light condition of 15 h/d for culture. About 15-25 days, a green dot grows. The seedlings were further differentiated after 30-40 days. When the buds of the resistant callus differentiated to about 2 cm, the seedlings were transferred to a rooting medium, cultured for about two weeks, and transplanted into the soil at room temperature. The coefficients of the TO-transgenic rice plants obtained by each structure were identified as shown in Table 1.

Table 1 TO-transgenic rice lines obtained from different structures

Example 8. Two-way gene promoter; ?SC/2 and;^ and Gt/S gene expression binary vector transformation into Arabidopsis thaliana.

1. A binary vector transformed into Agrobacterium tumefaciens

The bidirectional gene promoters 3⁄4SC 2 and ; CZ?, CaMV35S and Gt/S gene expression binary vectors (same as in Example 7) were electroporated into Agrobacterium strain GV3101 (Koncz C, Schell J. 1986. Mol Gen). Genet, 204:383-396, available from GAMBIA) Competent cells (competent cell preparation and electroporation were performed according to the method of Molecular Cloning (Sambrook et al, 1998)), cultured at 28 °C Single colony. The transformed Agrobacterium was inoculated into YM liquid medium containing 5 (^g/ml Kanamycin, shaken at 28 °C, 220 rpm for 16 hr, and the bacterial liquid was directly identified by PCR.

2. Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana

Arabidopsis thaliana transformation takes direct infiltration. Arabidopsis tMiana, the ecological Landsberg erecta, The European Arabidopsis Stock Centre (NW20), was selected to remove the knotted and unflowered flower buds. The correct Agrobacterium was identified by PCR in LB liquid medium (containing 50 g/ml Kanamycin and 100 g/ml rifampicin) to a OD _{6 (M} ) of about 0.6, and the cells were collected by centrifugation at 4000 g. The cells and the infiltrate (MS basic component + 5% sucrose, 30 μΙ/L of Titon 100) were uniformly mixed at 1:1. The mixed infiltrate was placed in a petri dish, and the flowering Arabidopsis flowers were fully infiltrated into the mixed infiltrate for 20-30 seconds. The excess liquid was removed by absorbent paper, moisturized for 2 days in the dark, and transferred to 21 'C light culture. . Mature genetically modified seeds (T0 Generation) mixed. The harvested T1 seeds were vortexed with 10 volumes of 10% sodium hypochlorite solution (containing 0.1% Ttiton 100) for 15 minutes, then rinsed 10 times with 10 volumes of sterile distilled water, all placed in 50 μ _§ / ml. On the MS plate of hygromycin, 4 Ό dark for 60 hours, transferred to 21 Ό light culture. After 14 days, the hygromycin-resistant plants were transplanted to the pots and continued to be cultured. After the seeds are mature, the individual plants are harvested, and the T1 transgenic lines are obtained. The coefficients of T1 transgenic Arabidopsis thaliana obtained by each structure were identified as follows: 24 strains of pSCU strain, 18 strains of ^ ^C strain, 16 strains of 0? 35S strain and 21 strains of WP strain.

Example 9. Identification and Analysis of GUS Activity in Transgenic Rice and Arabidopsis

1. Preparation of materials: For transgenic rice, the harvested TO-transgenic seeds (ie T1 generation) should be immersed in 30~50 waters per plant for 48 hours, and then transferred to 0.6% agar plates after seed germination (containing 50μ _§ /ml of hygromycin) on the germination. After 3 to 4 days, the seed rooting condition was investigated, and the normal rooting (ie, hygromycin-resistant) seedlings on the hygromycin plate were transferred to the field, which was the T1 generation transgenic seedling. Treat the T1 with the seedlings growing to 4 leaves and one heart. At the time of treatment, 10 lines were randomly selected for each structure, and 15 seedlings per line were divided into 3 groups: The first group was inoculated with M. oryzae strain 007, and the conidia were made to a concentration of 10 ⁵ with 0.025% Tween 20 aqueous solution. /ml of the suspension, sprayed on the upper leaves of the seedlings, darkened for 24 hours after inoculation at 27'C, and then continued to light, 36 hours after inoculation, inoculated leaves, frozen in liquid nitrogen, stored in -80 Ό refrigerator spare (sample Recorded as Inoculation); the second group of seedlings only sprayed 0.025% T _We en20 aqueous solution, the other treatment and sampling were the same as the first group (sample is Mock); the third group of seedlings without any treatment, with the first and second groups At the same time, the upper leaves of the healthy seedlings in the field were taken, frozen in liquid nitrogen, and stored in a -80 Ό refrigerator (sample is unter _ea ted). The above samples were used for quantitative analysis of GUS expression activity. GUS tissue staining material is the direct selection of roots, stems, leaves and sheaths of healthy seedlings.

For transgenic Arabidopsis, each harvested T1 seed is treated with 10 volumes of 10% sodium hypochlorite solution (including

0.1% Ttiton 100) was shaken for 15 minutes, then rinsed 10 times with 10 volumes of sterile distilled water, placed on MS plates containing 50 μml of hygromycin, 4 Ό dark for 60 hours, transferred to 21 Ό light culture . When the seedlings grow to 3-4 leaves, carefully select the leaves and roots. Ten lines were selected per structure, 20-30 per line. The selected tissue liquid nitrogen was quickly frozen and stored in a -80 ° C refrigerator for quantitative analysis of GUS expression activity. GUS tissue staining directly selected seedlings for whole plant staining.

2. Quantitative analysis of GUS expression activity: GUS activity assay was performed according to the method of Yang et al. (Yinong Yang et al, the Plant Journal, 2000, 22(6): 543-551). Specifically: take the tissue to be tested and rapidly grind it into liquid nitrogen, add appropriate amount of extraction buffer, grind into homogenate in ice bath, centrifuge at 12000 g for 10 minutes at 4 °C; take 5 μl of supernatant to add 45 μΐ 2 mM 4-MUG(4-methyl mbeUiferyl-D-glucuronide), and quickly pipet 5 μΐ of the mixture into Na ₂ C0 _{3 of} ΙΟΟμΙ 0.2 终止 to terminate the reaction as a control. Remaining mixture 37 ° C insulation After 30 min, 5 μl of the solution was added to ΙΟΟμΙ 0.2 Μ [Na ₂ C0 _{3 to} terminate the reaction. After the end of the reaction, the fluorescence value of the product 4-MU was measured on a TECAN GENios Plus fluorescent plate reader. At the same time, 5 μΐ of the supernatant was used to determine the total protein content by the Bradford method using a TECAN GENios Plus fluorescent plate reader. Then, the mass of 4-MU produced per minute per mg of protein was converted according to the dilution ratio, and the unit was nmol/min/mg protein, and the average value of 10 lines was calculated. The results are shown in Figures 8-13 and Figures 16, and Π.

According to the results of GUS activity, the following conclusions can be drawn: 1) Both _P SCI2 and pSCI3 have strong O. oxysporum-inducible promoter activity in transgenic rice leaves, and their activity in inoculation with Magnaporthe oryzae for 36 hours is CaMV35S. The promoters were 28-fold and 12-fold, respectively, by about 20-fold compared to the 0.025% Tween water spray control (Figures 8, 9, 10, 11 and 12). 2) The deletion of pSCI2-l after deletion of 69 bp at the 5' end was significantly reduced in rice induced by rice blast infection (Fig. 9); similarly, the 5' end of pSCB lacked 98 bp after pSCI3-I The expression activity induced by Magnaporthe oryzae infection was also significantly reduced (Fig. 10). This result indicates that the 5' end sequence is required for its full promoter activity. Since pSCU and pSCB are two complementary single strands of the same DNA fragment, it can be judged based on the above results: ^CJ2 or ^ is a promoter with a bidirectional driving gene expression ability in rice, which is infested by pathogens in leaves. . 3) j^C/2 and pSCJ3 have strong constitutive expression activities in roots and leaves of transgenic Arabidopsis thaliana. Their average activity in driving GUS expression at roots was 1.7-fold and 1.3-fold, respectively, of CaMV 35S (Fig. 15); the activity of driving GUS expression in leaves was 4 and 2.3 times that of CaMV 35S, respectively (Fig. 16). This result indicates that the bidirectional promoter can strongly constitutively drive the target gene in the roots and leaves of the dicotyledonous Arabidopsis thaliana, and can be used for genetic engineering of dicotyledons. 4) The 5' untranslated region of the OsSCI2 and OsSCI3 genes, the sequences of which are shown in SEQ ID NO: 5 and SEQ ID NO: 6 (containing an intron region, respectively), when SEQ ID NO: 5 is fused downstream of the pSCU (pSC/2+/), SEQ ID NO: 6 was fused to pSCI3 downstream ipSCI3+I, and post-driven reporter gene GUS, which was significantly enhanced by M. oryzae-inducible expression activity (Fig. 11 and Fig. 12). This result indicates that the 5' untranslated region has the ability to enhance gene expression as an enhancer. 5) Quantitative analysis of GUS gene expression by various derivative variants of the bidirectional gene promoter revealed that the GUS activity of PSCI3-3 transgenic plants was significantly higher than that of PSCI3-2 transgenic plants (Fig. 10), indicating pSCU-2 and There is a positively regulated cis element between pSCB^ which is between 213 and 400 bp in sequence SEQ ID NO: 2. 6) The deletion of the sequence of pSC (shown as SEQ ID NO: 1) from position 162 to position 932 drives the GUS reporter gene pSCISP2 or the sequence of ? (the sequence of which is shown in SEQ ID NO: 2) from position 162 to The 932 deletion drives the Gt« reporter gene ( _P SCISP3), and their transgenic plants both express the constitutive expression of GUS leaves and the enhanced induction of M. infestans infection (Fig. 13). Since the sequences of pSCISP2 and pSCISP3 are reverse complementary sequences, Therefore, it was judged that: ^OS 2 p5aSP3 has the ability to drive gene expression in both directions, which is constitutively expressed in rice leaves and has the characteristic of enhancing the infection of pathogenic bacteria.

3. GUS tissue staining: GUS tissue staining was performed according to Jefferson et al. (Jefferson, et al., EMBO J., 1987, 6: 3901-3907). The selected transgenic material was first cleaned on a sterile water surface. After blotting with sterile absorbent paper, immersed in GUS staining solution, pumping for 5 minutes, then placing at 28 ° C, shaking on a 200 rpm shaker for 30 minutes to overnight, and then bleaching in 70% or 100% ethanol to develop color. The results showed that both the bidirectional gene promoter and its derived variants could drive genes induced by rice blast fungus in the leaves of transgenic rice (Fig. 14), but constitutively expressed in the roots of healthy transgenic rice, but the expression was strong. Slightly different. Its characteristic at the root is expressed mainly in the apical meristem and lateral root germination (Fig. 15). The bidirectional gene promoter drives the Gi/S gene to be constitutively expressed in the leaves of the transgenic Arabidopsis roots (Fig. 18). Example 10. Construction of disease-resistant binary vectors

This binary vector was constructed on the basis of Example 4. Here, only the construction process of the disease-resistant binary vector derived from pSCI2 is taken as an example, and other derivative variants of a series of promoters can be obtained by this method. The plasmid was digested with BstE II for 6 hours, then filled in with T4 DNA polymerase, phenol extracted and dissolved in double-distilled sterile water, digested with Nco l, and the vector fragment was recovered to link the endogenous or foreign gene to be chimeric. II, which may have an existing or later increased N^I site linker at the 5' end, and its 3' end should be a smooth end. To link the endogenous or exogenous gene I, the SC plasmid can be digested with EcoRI, filled in, then ligated into the blunt-ended gene of interest, and the direction of ligation is determined. In addition, in order to be able to chise various genes, you can use Nco: [double-enzyme-cutting, blunt-ended, ready-to-chimeric gene II, and identify the direction of ligation, and then digest the plasmid with EcoRI. Fill in, then connect to the blunt-ended gene of interest I (Figure 6).

Claims

Claim

A bidirectional gene promoter, a variant thereof, or a functional equivalent thereof, the promoter having the sequence of SEQ ID NO: 1 or SEQ ID O.-2; the variant having SEQ ID NO: 1 a sequence resulting from one or more deletions, additions, substitutions or modifications of bases 162 to 932 in the sequence shown, or having positions 217 to 987 in the sequence shown in SEQ ID NO: The base is subjected to one or several deletions, additions, substitutions or modifications; the functional equivalent has the sequence shown in any one of SEQ ID NOS: 7-13, and the TATA cassette is fused at the 5' end.

2. The bidirectional gene promoter of claim 1, the sequence of which is SEQ ID NO: 1, 2, 3, 4, 14 or

15 is shown.

A promoter having the sequence shown in any one of SEQ ID NOS: 7-13.

An enhancer having the sequence shown in any one of SEQ ID NOS: 5, 6.

5. An isolated DNA as a key cis-acting element having the sequence shown in any one of SEQ ID NOS: 16, 17.

A recombinant nucleic acid sequence comprising the promoter of any one of claims 1 to 3.

7. The recombinant nucleic acid sequence of claim 6, further comprising at least one functional endogenous or exogenous gene. ,

8. The recombinant nucleic acid sequence of claim 7, wherein the functional endogenous or exogenous gene is selected from the group consisting of a disease resistance gene, a defense gene and related genes isolated from a plant, a key enzyme or protein in a defense process. Gene, a phytoalexin synthase gene having direct or indirect bactericidal action; an avirulence gene isolated from rice blast fungus or an antisense RNA of a gene closely related to rice blast fungus, an animal or a microorganism isolated from disease resistance Genes of active substances or genes that synthesize their enzymes, and stress resistance genes that are resistant to drought, cold, salt or high temperature.

9. A recombinant construct comprising the recombinant nucleic acid sequence of any of claims 6-8.

10. An expression system comprising the recombinant construct of claim 9.

11. Use of a promoter according to any one of claims 1 to 3 for driving expression of an endogenous or exogenous gene in a plant.

12. The use of claim 11, wherein the plant is a monocot or a dicot.

13. The use of claim 11, wherein the monocot is rice and the dicot is Arabidopsis.

A plant cell comprising the recombinant nucleic acid sequence of any one of claims 6-8.

15. A method of obtaining a transgenic plant comprising transforming the expression system of claim 10 into a plant.

16. A method for isolating a two-way gene promoter comprising cDNAs of a plurality of genes isolated and expressed from an organism; analyzing the specific positions of the cDNAs on the chromosome; and finding two connected sides on a piece of DNA, And the cDNA with the opposite transcription direction; by transgenic analysis and confirmation whether the DNA of a certain segment is a bidirectional gene promoter.

17. The method of claim 16 wherein the organism is rice or Arabidopsis.

18. A method for isolating the promoter of any one of claims 1 to 3, comprising: (1) preparing a rice cDNA library infected by rice blast fungus, and plating the membrane; (2) using healthy rice leaves The mRNA is hybridized with the probe, and the unhybridized clone is selected as the candidate inducible cDNA clone and confirmed by reverse Northern; (3) the confirmed inducible cDNA sequence is compared with the published rice genome database to find 5, Two cDNAs that are opposite and matched to a complementary strand of a DNA fragment; (4) The bidirectional gene promoter function of the DNA fragment is verified by detecting the expression of the reporter gene by ligating the DNA fragment to a reporter gene.