CN107129990B - Application of Inhibition or Mutation of TFIIAγ Gene in Crop Disease Resistance - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering, in particular to the application of inhibiting or mutating TFIIAγ gene in crop disease resistance. The genes comprise rice OsTFIIAγ5 gene, pepper CaTFIIAγ gene h and tomato SlTFIIAγ gene, and the invention relates to functional verification in the interaction between plants and bacterial pathogens carrying transcriptional activator-like effector (TALE) genes. TFIIAγ gene is a negative regulator in plant resistance to TALE-carrying pathogens. Inhibiting the expression of OsTFIIAγ5 gene or mutating this gene can make rice have broad-spectrum resistance to bacterial blight and bacterial leaf spot. However, the mutant OsTFIIAγ5 gene did not affect the yield traits of rice. Inhibition of SlTFIIAγ gene expression in tomato increases tomato resistance to bacterial scab. Inhibition of CaTFIAγ gene expression in pepper can improve the resistance of pepper to spot disease.

Description

Application of Inhibition or Mutation of TFIIAγ Gene in Crop Disease Resistance

technical field

The invention belongs to the technical field of plant genetic engineering. Specifically, it relates to the application of inhibiting or mutating TFIIAγ gene in crop disease resistance. The application relates to the application of plant transcription factor IIAgamma subunit (TFIIAγ) gene in bacterial disease resistance. TFIIAγ gene is a negative regulator in disease resistance response. Plants that inhibited the expression of TFIIAγ gene significantly improved their ability to carry transcription activator-like effector (TALE) pathogens.

Background technique

Xanthomonas (Xanthomonas) is a gram-negative genus of prokaryotic Pseudomonas family, all Xanthomonas bacteria are plant pathogens, it includes many pathogenic species, is a worldwide pathogenic bacteria , can harm more than 400 types of plants in at least 27 plant genera (Ryan et al. 2011). It brings serious harm to the safe production of various plants every year, not only harming important food crops, but also seriously harming important economic crops and vegetables, such as Xanthomonas oryzae pv. Xoo) and bacterial leaf spot (Xanthomonas oryzae pv. Oryzicola, Xoc) cause bacterial blight and bacterial leaf spot of rice, respectively; Xanthomonas campestris pv. vesicatoria (Xcv) can cause tomato Bacterial scab and pepper spot disease of tomato and pepper; citrus canker (Xanthomonas axonopodis pv.citri, Xac) can cause citrus canker; cotton horn spot (Xanthomonas campestris pv. malvacearum, Xcm) can cause cotton canker ; Cruciferous vegetable black rot fungus (Xanthomonas campestris pv.Armoraciae, Xca) can cause black rot disease of cruciferous vegetables; Cassava bacterial wilt fungus (Xanthomonas axonopodispv.manihotis, Xam) can cause cassava bacterial wilt disease; Banana bacteria Xanthomonascampestris pv.Musacearum can cause bacterial wilt disease of banana; Xanthomonascampestris pv.vasculorum can cause sugarcane slippery disease; Xanthomonascampestris pv. Glycines can cause soybean spot disease (Schornack et al. 2006; Ryan et al. 2011; Bart et al. 2012).

Xanthomonas genus harms plants mainly by secreting its own transcription activator-like effector (TALE) into the nucleus of plant cells through the type III secretion system after infecting plant cells. Different pathogenic species of Xanthomonas carry different numbers of TALEs, and these TALEs are usually directly combined with a specific region of the promoter region of the target gene to achieve the purpose of making the host pathogenic by activating and inducing the expression of the target gene in the host ( Scholze and Boch 2011).

During the transcription of eukaryotic mRNA, the most characterized core promoter unit is the TATA box, which is first recognized by the basic transcription factor TATA box-binding protein, and then other basic transcription factors such as TFIIA (basal transcription factor IIA) and TFIIB. The sequence binds to it to form the pre-initiation complex of transcription. TFIIA interacts with TATA box-binding proteins, making TATA box-binding proteins more stably bound to the TATA box to initiate gene transcription into mRNA (Yudkovsky et al. 2000).

Transcriptase II-dependent transcription in eukaryotes requires TFIIA. TFIIA consists of three subunits, α, β, and γ, which are encoded by two genes; one gene encodes TFIIAαβ, and the other gene encodes TFIIAγ (Li et al. 1999). The amino acid sequences of TFIIAγ derived from different eukaryotes are highly similar. A TFIIAγ gene in rice is located on chromosome 5 and named OsTFIIAγ5. The encoded OsTFIIAγ5 consists of 106 amino acids. When the 39th amino acid is mutated from valine to glutamic acid, it is xa5, which is recessive. The major gene for bacterial blight resistance is encoded by xa5 (Iyer and McCouch 2004; Jiang et al. 2006).

At present, it is known that Xanthomonas bacteria containing transcriptional activator-like effectors infect their host plants, secrete TALE into the host cells and bind to specific regions of the host's target disease-susceptibility gene promoter to activate the host's disease-susceptible gene expression, causing disease (Boch et al. 2014). But whether other proteins are involved in this process, or how the host's essential transcription factors play a role in this process, is unclear.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects existing in the prior art, and to inhibit or mutate the application of TFIIAγ gene in crop disease resistance. The present invention finds that the different TALEs of Bacterial blight and Bacterial leaf spot can bind to OsTFIIAγ5 of the host rice, but weaken the binding to xa5. Rice carrying the recessive xa5 gene not only developed broad-spectrum resistance to bacterial blight, but also developed broad-spectrum resistance to bacterial leaf spot.

Further, the present invention finds that inhibiting the expression of the OsTFIIAγ5 gene in rice can enhance the broad-spectrum resistance of transgenic rice to bacterial blight and bacterial leaf spot; inhibiting the expression of the CaTFIIAγ gene in pepper can enhance the resistance to pepper spot; The expression of SlTFIIAγ gene in tomato can enhance the resistance to tomato bacterial scab.

The invention relates to genetic manipulation of cDNA fragments of TFIIAγ genes of different plants to identify their functions. The fragments endow rice with disease resistance to diseases caused by bacterial blight and bacterial leaf spot, and endow peppers with resistance to diseases caused by pepper spot pathogens. The disease produced a disease resistance response, giving tomato a disease resistance response to the disease caused by the tomato bacterial scab fungus. The protein sequences of the isolated TFIIAγ genes of rice, pepper and tomato are shown in SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9 in the sequence listing, or substantially equivalent to SEQ ID NO: 5, SEQ ID NO: : 7 and the protein sequence shown in SEQ ID NO: 9. Analysis of their sequences revealed that they are a class of transcription factors. Inhibiting the expression of the sequence shown in SEQ ID NO: 4 can enhance the resistance of rice to Bacterial blight and bacterial leaf spot; inhibiting the expression of the sequence shown in SEQ ID NO: 6 can enhance the resistance of pepper to Capsicum speckle; inhibiting SEQ ID NO: 6 The expression of the sequence shown in ID NO: 8 can enhance the resistance of tomato to Tomato bacterial scab,

In the Examples section of the present invention, the applicant described the genetic manipulation, functional verification and application process on rice, pepper and tomato, and the characteristics of the above three genes.

Description of drawings

SEQ ID NO: 1 is the nucleotide sequence of the rice OsTFIIAγ5 gene isolated and cloned by the present invention, and the sequence length is 6261 bp. The 5'UTR segment is 1-374, and the length is 374bp; the first exon segment is 375-503, and the length is 129bp; the second exon segment is 598-648, and the length is 51bp; the third exon segment is 5744-5884, the length is 141bp; the 3' UTR segment is 5885-6261, the length is 377bp; the first intron segment is 504-597, The length is 94bp; the second intron segment is 649-5743 and 5095bp in length.

SEQ ID NO: 2 is the nucleotide sequence of the pepper CaTFIIAγ gene isolated and cloned by the present invention, and the sequence length is 5695 bp. Among them: the first exon segment is 1-129, the length is 129bp; the second exon segment is 2451-2501, the length is 51bp; the third exon segment is 5555-5695 The first intron segment is 130-2450 bp and the length is 2321 bp; the second intron segment is 2502-5554 bp and the length is 3053 bp.

Sequence Listing SEQ ID NO: 3 is the nucleotide sequence of the tomato SlTFIIAγ gene isolated and cloned in the present invention, and the sequence length is 5028 bp. Among them: the first exon segment is 1-129, the length is 129bp; the second exon segment is 2265-2315, the length is 51bp; the third exon segment is 4888-5028 The first intron segment is 130-2264 bp and the length is 2135 bp; the second intron segment is 2316-4887 bp and the length is 2572 bp.

Sequence Listing SEQ ID NO: 4 is the coding sequence (CDS) of the spliced rice OsTFIIAγ5 gene.

Sequence Listing SEQ ID NO: 5 is the protein sequence of the rice OsTFIIAγ5 gene.

Sequence Listing SEQ ID NO: 5 is the coding sequence (CDS) of the spliced pepper CaTFIAγ gene.

Sequence Listing SEQ ID NO: 7 is the protein sequence of the pepper CaTFIAγ gene.

SEQUENCE LISTING SEQ ID NO: 8 is the coding sequence (CDS) of the spliced tomato SlTFIIAγ gene.

Sequence Listing SEQ ID NO: 9 is the protein sequence of the tomato SlTFIIAγ gene.

Figure 1. The flow chart of the present invention to isolate and clone TFIIAγ gene from rice, tomato and pepper and to verify the function of TFIIAγ gene.

Figure 2. Structure of the OsTFIIAγ5 gene in rice, tomato and pepper. Description of reference numerals: black rectangles indicate exons, black horizontal lines indicate introns, and white rectangles indicate 5' and 3' untranslated regions (UTRs). "ATG" and "TAA" are translation initiation and termination codons, respectively. The numbers indicate the number of nucleotides in each structure. Arrows indicate the position and orientation of PCR amplification primers 1F, 1R, gF, gR, dsF and dsR.

Figure 3. Structure of the genetic transformation vector pDS1301 carrying the OsTFIIAγ5 gene. It is an inverted repeat of the cDNA fragment of the OsTFIIAγ5 gene inserted into the multiple cloning site of the pDS1301 vector. Description of reference numerals: RB and LB represent T-DNA right and left borders; Hpt represents hygromycin phosphotransferase gene (screening gene); 35S represents cauliflower mosaic virus promoter; AdhI represents maize alcohol dehydrogenase gene inclusion Sub I; Waxy-a represents rice Waxy gene intron; OCS represents octopine synthesis acid gene terminator; GUS represents β-glucuronidase gene (marker gene).

Figure 4. OsTFIIAγ5 gene expression and response to bacterial blight strain PXO99 in T0 generation genetically transformed plants. The control was the rice variety Zhonghua 11 (the genetically transformed recipient). The expression level of OsTFIIAγ5 gene in genetically transformed plants is relative to the expression level of OsTFIIAγ5 gene in control Zhonghua 11.

Figure 5. Co-segregation of OsTFIIAγ5 gene expression and phenotype in T1-generation genetically transformed plants suppressing OsTFIIAγ5 gene expression. The control was the rice variety Zhonghua 11 (the genetically transformed recipient). The expression level of OsTFIIAγ5 gene in genetically transformed plants is relative to the expression level of OsTFIIAγ5 gene in control Zhonghua 11.

Fig. 6. Plants of T1 generation genetically transformed families OsTFIIAγ5-RNAi1, OsTFIIAγ5RNAi-3 and OsTFIIAγ5-RNAi7 were inoculated with 8 species of bacterial blight of the Philippines, 1 species of bacterial blight of Japan, and 1 species of bacterial blight of Korea Phenotypes of physiological races and four species of bacterial blight species after two weeks.

Fig. 7. Phenotypes of the T1 generation genetically transformed lines OsTFIIAγ5-RNAi1, OsTFIIAγ5-RNAi3 and OsTFIIAγ/5-RNAi7 plants inoculated with 5 strains of Chinese bacterial leaf spot two weeks later.

Figure 8. Rice cultivars carrying recessive xa5 (encoding xa5 or mutant OsTFIIAγ5 and rice cultivars carrying OsTFIIAγ5 were inoculated with 9 bacterial races of Philippine bacterial blight, 1 physiological race of Japanese bacterial blight, and 1 Korean white leaf Phenotypes of B. blight races and 4 species of B. solani after two weeks.

Figure 9. Phenotypes of rice cultivars carrying recessive xa5 (encoding xa5 or mutant OsTFIIAγ5 and rice cultivars carrying OsTFIIAγ5 and rice cultivars carrying OsTFIIAγ5 two weeks after inoculation with 5 Chinese bacterial races.

Figure 10. Structure of the transformation vector pTRV2-SlTFIIAγ carrying the tomato SlTFIIAγ gene. It is a part of the sequence of the cDNA fragment of the tomato SlTFIIAγ gene inserted into the multiple cloning site of the pTRV2 vector. Description of reference numerals: RB and LB represent T-DNA right and left borders; 35S represents cauliflower mosaic virus promoter; CP represents tobacco chipping virus capsid protein; Rz represents self-cleaving ribozyme; Waxy-a represents rice Waxy Gene intron; NOSt stands for nopaline synthase gene terminator.

Figure 11. Inhibition of SlTFIIAγ gene expression in tomato and response to tomato bacterial scab 23-1 by gene silencing technology induced by tobacco rattle virus. The control was the suppression of the green fluorescent protein GFP gene in tomato by using the gene silencing technology induced by tobacco chipping virus. Panel A in Figure 11 shows that inhibiting the expression of SlTFIIAγ gene in tomato can enhance the resistance of tomato to tomato bacterial scab 23-1; Panel B in Figure 11 is the use of tobacco chip virus-induced gene silencing technology to inhibit tomato SlTFIIAγ gene expression detection results; Figure C in Figure 11 shows the bacteria in the leaves on the 0th and 3rd day after inoculation of tomato bacterial scab 23-1 after suppressing the SlTFIIAγ gene of tomato by the gene silencing technology induced by tobacco crisp virus number of growths.

Figure 12. Structure of the transformation vector pTRV2-CaTFIIAγ carrying the pepper CaTFIAγ gene. It is a part of the sequence of the cDNA fragment of the pepper CaTFIIAγ gene inserted into the multiple cloning site of the pTRV2 vector. RB and LB represent the right and left borders of T-DNA; 35S represents the cauliflower mosaic virus promoter; CP represents the capsid protein of tobacco Rattle virus; Rz represents the self-cleaving ribozyme; Waxy-a represents the rice Waxy gene intron; NOSt stands for nopaline synthase gene terminator.

Fig. 13. Inhibition of CaTFIIAγ gene expression in pepper and response to pepper spot disease 23-1 by tobacco rattle virus-induced gene silencing technology. The control is the suppression of the green fluorescent protein GFP gene in pepper by using the gene silencing technology induced by tobacco crisp virus. Panel A in Figure 13 shows that inhibiting the expression of CaTFIIAγ gene in pepper can enhance the resistance of pepper to pepper spot disease 23-1; Panel B in Figure 13 is the inhibition of CaTFIIAγ gene in pepper by gene silencing technology induced by Tobacco Rattle virus Expression detection results; panel C in Figure 13 is the number of bacterial growth in leaves on day 0 and day 3 after inoculation of pepper spot disease 23-1 after suppressing the CaTFIAγ gene of pepper by using tobacco chipping virus-induced gene silencing technology.

Detailed ways

The present invention is further defined in the following examples. Figure 1 describes the process of isolating and cloning rice OsTFIIAγ5 gene and verifying the function of rice OsTFIIAγ5 gene, and the process of isolating tomato SlTFIIAγ gene and pepper CaTFIIAγ gene and verifying the function. From the above description and the following examples, those skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the invention, can make various changes and modifications of the present invention to make it applicable Various uses and conditions.

Example 1: Sequence and structural analysis of the TFIIAγ gene

1. Prediction of TFIIAγ gene structure

The applicant used a DNA sequence (2944bp～3345bp) of a TALE gene pthXo1 (GenBank accession number: ACD58243) of bacterial blight PXO99 as a bait protein to screen the cDNA library of rice variety Zhonghua 11 (Yuan et al., 2010), and screened the A cDNA sequence was searched in the nucleotide database GenBank (http://www.ncbi.nlm.nih.gov) by the BLAST method (Altschul et al. 1997), and it was found that the cDNA sequence was related to a cDNA sequence from the rice variety Nipponbare located on rice chromosome 5. The sequences are highly homologous. In the rice genome database TIGR (http://rice.plantbiology.msu.edu/), this rice sequence is annotated as a gene whose number is LOC_Os05g01710, which is the gene OsTFIIAγ5 involved in the present invention.

2. Isolation and cloning of OsTFIIAγ5 gene from rice variety Zhonghua 11

Rice Zhonghua 11 is a rice variety commonly used in scientific research (Cao et al., 2007). The applicant designed two PCR primers, TFIIAγgF (5′-GGCGTAAAATAAAGAGAAATCTGG-3′) and TFIIAγgR, according to the position and structure of the OsTFIIAγ5 gene in the japonica rice Nipponbare genome (rice genome database TIGR) and the genome sequences on both sides of the OsTFIIAγ5 gene as templates. (5'-TAATGTAAAGGTACCATTACATAGG-3'), a DNA fragment of 6261 bp in full length was amplified from Zhonghua 11 containing the OsTFIIAγ5 gene. Both ends of the PCR amplified fragments were sequenced by the dideoxynucleotide end-termination method (Applied Biosystems, USA) using a sequencing kit from Applied Biosystems, USA. A DNA sequence with a length of 6261 bp was obtained after the sequence was assembled, which included the complete OsTFIIAγ5 gene sequence.

3. Analysis of OsTFIIAγ5 gene structure

In order to obtain the full-length cDNA sequence of the OsTFIIAγ5 gene, the applicant extracted the total RNA of Hua 11 in the rice variety (Zhou et al. 2002). Take 1-5 μg total RNA and treat it with DNaseI (Invitrogen, USA) for ₁₅ minutes to remove genomic DNA contamination, and then refer to the method of Zhou et al. Promega Corporation, USA) was reverse transcribed into cDNA. Then, using PCR primers TFIIAγcDNAF (5′-CCGGAATTCATGGCCACCTTCGAGCTC-3′) and TFIIAγcDNAR (5′-CGCGGATCCTTATTGGCTGAGTAGTTTG-3′), the full-length cDNA of OsTFIIAγ5 gene was amplified with the total cDNA as a template. Then the PCR product is connected with the TA cloning vector pGEM-T (Promega, USA), and the connected vector is electro-transformed (the electro-transformer is a product of eppendorf company, the voltage used in this example is 1800V, and the specific operation refers to the instruction manual of the instrument ) into E. coli strain DH10B (Sun et al., 2004), and positive clones were screened by enzyme digestion. The positive clones were sequenced to obtain the cDNA sequence of the OsTFIIAγ5 gene (see SEQ ID NO: 1).

Comparative analysis of the genome sequence and cDNA sequence of the OsTFIIAγ5 gene showed that the OsTFIIAγ5 gene was composed of 6261 nucleotides (see SEQ ID NO: 1), including three exons and two introns (see Figure 2). The first exon consists of 129 nucleotides (located at positions 375-503 of SEQ ID NO: 1 in the sequence listing), 5'UTR (located at positions 1-374 of SEQ ID NO: 1 in the sequence listing); the first The intron consists of 94 nucleotides (located at positions 504-597 of SEQ ID NO: 1 of the sequence listing); the second exon consists of 51 nucleotides (located at 598 of SEQ ID NO: 1 of the sequence listing). -648); the second intron consists of 5095 nucleotides (located at positions 649-5743 of SEQ ID NO: 1 of the sequence listing); the third exon consists of 141 nucleotides (located in the sequence Listed at positions 5744-5884 of SEQ ID NO: 1, the 3'UTR is located at positions 5885-6261 of SEQ ID NO: 1 in the sequence listing, and consists of 377 nucleotides.

4. Analysis of the encoding product of OsTFIIAγ5 gene

The coding region of OsTFIIAγ5 gene consists of 321 nucleotides and encodes a protein with a length of 106 amino acids. This protein is a transcription factor.

5. Isolation and cloning of SlTFIIAγ gene from tomato and analysis of gene structure

The applicant searched the tomato gene sequence in the international public nucleotide database according to the rice OsTFIIAγ5 gene, and designed two PCR primers, SlTFIIAγF (5′-CGCGGATCCATGGCGACTTTTGAGCTATAC-3′) and SlTFIIAγR (5′-CCGCTCGAGTCACTGTGTGAGCAGCTTTGAG-3′). A complete cDNA fragment containing the tomato SlTFIIAγ gene was amplified from the cDNA obtained by reverse transcription of the total RNA of the tomato variety Ailsa Craig, but not limited to this variety). Both ends of the PCR amplified fragments were sequenced by the dideoxynucleotide end termination method (Applied Biosystems, USA) using a sequencing kit from Applied Biosystems, USA. A DNA sequence with a length of 321 bp was obtained after sequence splicing, which included the complete cDNA sequence of the SlTFIIAγ gene. Using PCR primers SlTFIIAγVF (5'-CCGGAATTCTTTGACAAGTCAATGACTG-3') and SlTFIIAγVR (5'-CGCGGATCCTCACTGTGTGAGCAGCTTTG-3'), a partial cDNA sequence containing 201 bp was PCR amplified from the clone containing the complete tomato SlTFIIAγ gene cDNA sequence. According to the position and structure of the SlTFIIAγ gene in the tomato genome, the applicant designed two PCR primers, SlTFIIAγgF (5′-ATGGCGACTTTTGAGCTATACAG-3′) and TFIIAγgR (5′-TCACTGTGTGAGCAGCTTTGAG-3, using the genome sequences on both sides of the SlTFIIAγ gene as templates) '), a 5028 bp DNA fragment containing the SlTFIIAγ gene was amplified from AilsaCraig. Both ends of the PCR amplified fragments were sequenced by the dideoxynucleotide end-termination method (Applied Biosystems, USA) using a sequencing kit from Applied Biosystems, USA. A DNA sequence with a length of 5028bp was obtained after the sequence was assembled, which included the complete SlTFIIAγ gene sequence. Comparing and analyzing the genome sequence and cDNA sequence of the SlTFIIAγ gene, it was determined that the SlTFIIAγ gene was composed of 5028 nucleotides, including three exons and two introns (Fig. 2). The first exon consists of 129 nucleotides (located at positions 1-129 of SEQ ID NO: 3 of the sequence listing); the first intron consists of 2135 nucleotides (located in SEQ ID NO: 3 of the sequence listing) 3 of 130-2264); the second exon consists of 51 nucleotides (located at 2265-2315 of SEQ ID NO: 3 of the sequence listing); the second intron consists of 2572 nucleotides (located at positions 2316-4887 of SEQ ID NO: 3 of the Sequence Listing); the third exon consists of 141 nucleotides (located at positions 4888-5028 of SEQ ID NO: 3 of the Sequence Listing).

6. Isolation and cloning of CaTFIIAγ gene from pepper and analysis of gene structure

The applicant searched the pepper gene sequence in the international public nucleotide database according to the rice OsTFIIAγ5 gene, and designed two PCR primers, CaTFIIAγF (5′-CGCGGATCCATGGCGACTTTTGAGCTATAC-3′) and CaTFIIAγR (5′-CCGCTCGAGTCACTGTGTGAGCAGCTTTGAG-3′). A complete cDNA fragment comprising the pepper CaTFIAγ gene was amplified from the cDNA obtained by reverse transcription of the total RNA of pepper variety Hua 50, but not limited to this variety. Both ends of the PCR amplified fragments were sequenced by the dideoxynucleotide end termination method (Applied Biosystems, USA) using a sequencing kit from Applied Biosystems, USA. A DNA sequence with a length of 321 bp was obtained after the sequence splicing, which included the complete cDNA sequence of the CaTFIIAγ gene. Using the PCR primers CaTFIIAγVF (5′-CCGGAATTCTTTGACAAGTCAATGACTG-3′) and CaTFIIAγVR (5′-CGCGGATCCTCACTGTGTGAGCAGCTTTG-3′), a partial cDNA sequence containing 201 bp was PCR amplified from the clone including the complete pepper CaTFIIAγ gene cDNA sequence. The applicant designed two PCR primers, CaTFIIAγgF (5′-ATGGCGACTTTTGAGCTATACAGG-3′) and CaTFIIAγgR (5′-TCACTGTGTGAGCAGCTTTG-3, according to the position and structure of the CaTFIIAγ gene in the pepper genome and the genome sequences on both sides of the CaTFIIAγ gene as templates. '), a 5695 bp DNA fragment containing the CaTFIIAγ gene was amplified from the pepper variety Hua 50. Both ends of the PCR amplified fragments were sequenced by the dideoxynucleotide end-termination method (Applied Biosystems, USA) using a sequencing kit from Applied Biosystems, USA. A DNA sequence with a length of 5695bp was obtained after the sequence was spliced, which included the complete sequence of the CaTFIIAγ gene. Comparing and analyzing the genome sequence and cDNA sequence of the CaTFIIAγ gene, it was determined that the CaTFIIAγ gene consists of 5695 nucleotides, including three exons and two introns (see Figure 2). The first exon consists of 129 nucleotides (located at positions 1-129 of SEQ ID NO: 2 in the sequence listing); the first intron consists of 2321 nucleotides (located in SEQ ID NO: 2 in the sequence listing) 130-2450; the second exon consists of 51 nucleotides (located at 2451-2501 of SEQ ID NO: 2 of the Sequence Listing); the second intron consists of 3053 nucleotides (located at The third exon consists of 141 nucleotides (positions 5555-5695 of SEQ ID NO: 1 in the sequence listing).

Genome sequence comparison analysis showed that the cDNA sequences of tomato SlTFIIAγ gene and pepper CaTFIIAγ gene were identical, and they encoded the same protein. Their protein sequences are different from those of rice.

Example 2: Inhibiting the expression of rice OsTFIIAγ5 gene to verify its function in rice disease resistance

1. Construction of genetic transformation vector for inhibiting expression

The present invention adopts RNA interference (RNAi) technology (Smith et al., 2000) to verify the function of the gene by inhibiting the expression of the OsTFIIAγ5 gene in the rice variety Zhonghua 11. The main mechanism of this technical approach: Partial fragments of the target gene are ligated in the form of inverted repeats with a vector capable of expressing double-stranded RNA (dsRNA), and the vector is introduced into plants through genetic transformation. The obtained transformed plants express a large amount of dsRNA that is homologous to a partial fragment of the target gene. These dsRNAs rapidly form short interfering RNAs (siRNAs). These siRNAs are complementary to the target gene's transcript (mRNA) and degrade the target gene's transcript under the action of special enzymes in the cell, thereby inhibiting the function of the target gene at the mRNA level. Researchers can verify the function of the target gene by changing the phenotype of the transgenic plant. RNA interference technology has been widely used for the verification of gene function (Smith et al., 2000).

The present invention utilizes the Agrobacterium-mediated genetic transformation vector capable of expressing dsRNA is pDS1301 (Chu et al., 2006, Figure 3), and the cDNA fragment of the OsTFIIAγ5 gene is transformed into the rice variety Zhonghua 11 by the above-mentioned Agrobacterium-mediated genetic transformation method , inhibited the expression of OsTFIIAγ5 gene in Zhonghua 11. The specific operation steps are as follows:

The cDNA clone of TFIIAγ/Xa5 gene (Figure 2), the intermediate vector pGEM-G (Promega, USA) and the genetic transformation vector pDS1301 were digested with restriction enzymes BamHI and KpnI, and the restriction was inactivated in a water bath at 65 °C for 10 min after the digestion was complete. sex endonuclease. The enzyme-digested fragment was electrophoresed on a 0.8% agarose gel and the target band was recovered. The exogenous fragment of cDNA clone was ligated with intermediate vector pGEM-GZ and genetic transformation vector pDS1301 with T4-DNA ligase, respectively. The ligated vector was electro-transformed into Escherichia coli strain DH10B, and the cloned plasmid obtained after electro-transformation was screened by BamHI and KpnI to verify positive clones.

The intermediate vector plasmid carrying the exogenous fragment of the cDNA clone and the pDS1301 vector plasmid that had been ligated into the first-strand exogenous fragment were digested with restriction enzymes SacI and SpeI. After the digestion was complete, the restriction enzymes were inactivated in a water bath at 65 °C for 10 min. The exogenous fragment of the cDNA clone was ligated into the SacI/SpeI restriction site of the pDS1301 plasmid carrying the OsTFIIAγ5 fragment using T4-DNA ligase. The positive plasmid clones were screened by SacI and SpeI double digestion reaction, and the positive clone was named pDS1301-TFIIAγ (Fig. 3).

2. Genetic transformation and analysis of T ₀ generation genetically transformed plants

pDS1301-TFIIAγ was introduced into the rice cultivar Zhonghua 11 using the Agrobacterium-mediated genetic transformation method (Lin and Zhang, 2005). The obtained genetically transformed plant was named OsTFIIAγ5-RNAi. In the present invention, a total of 10 independently transformed plants are obtained. All transformed plants were inoculated with bacterial blight strain PXO99 at the adult stage (strain PXO99 was a gift from the International Rice Research Institute of the Philippines, Sun et al., 2004), compared with the control Zhonghua 11 and the negative plants of genetic transformation, 8 strains were positive for genetic transformation The resistance of the plants was significantly enhanced (P<0.01) (Table 1).

Table 1 Response of T0 generation genetically transformed plants (pDS1301-TFIIAγ) to bacterial blight strain PXO99

(1) Each genetically transformed gene plant was inoculated with 5-7 leaves, and the length of the lesions was investigated 14 days later, and each data was obtained from the average value of multiple leaves.

(2) Negative transformed plants, other plants are positive transformed plants.

To further verify whether the enhanced disease resistance of genetically transformed plants is related to the expression of OsTFIIAγ5 gene, the present invention uses the above real-time quantitative RT-PCR method to detect the expression of OsTFIIAγ5 gene in 10 T0 generation genetically transformed plants. The experimental results showed that the expression of OsTFIIAγ5 gene in transformed plants was significantly negatively correlated with the length of plant lesions (correlation coefficient was 0.8748, α=0.01, n=10) (Fig. 4). Compared with the control material Zhonghua 11, the expression of OsTFIIAγ5 gene in the genetically transformed plants with enhanced disease resistance was significantly decreased (P<0.01). However, the expression of OsTFIIAγ5 gene in negatively transformed plants with no obvious change in disease resistance phenotype had no significant change compared with Zhonghua 11 (P>0.05). The results indicate that the enhanced resistance of genetically transformed plants to bacterial blight is due to the decreased expression of OsTFIIAγ5 gene; the encoded product of OsTFIIAγ5 gene plays a role as a negative regulator in rice resistance to bacterial blight; inhibits the expression of OsTFIIAγ5 gene in rice Expression can enhance the resistance of rice to bacterial blight.

3. Analysis of T1 Generation Genetically Transformed Plants

The T1 generation lineages of two genetically transformed plants (OsTFIIAγ5-RNAi3 and OsTFIIAγ5-RNAi7) with enhanced resistance in the TO generation were analyzed. Bacterial blight PXO99 was inoculated in the field at the booting stage, and the specific PCR primers pMCG-f (5'-GGCTCACCAAACCTTAAACAA-3') and pMCG-r (5'-CTGAGCTACACATGCTCAGGTT-3') on the genetic transformation vector pDS1301 were used at the same time. Test positive plants. The results showed that all plants with significantly enhanced resistance compared with the control Zhonghua 11 carried the pDS1301 vector sequence; while the plants with no significant change in resistance compared with the control did not carry the pDS1301 vector sequence (Table 2, Figure 5). This indicated that the disease-resistant phenotype of the genetically transformed plants was co-segregated with the transfected OsTFIIAγ5 gene fragment that could form double-stranded RNA, further proving that inhibiting the expression of OsTFIIAγ5 could enhance rice disease resistance.

Table 2 Response of T1 generation genetically transformed plants (pDS1301-TFIIAγ) to bacterial blight strain PXO99

(1) Each genetically transformed gene plant was inoculated with 5-7 leaves. After 14 days, the diseased spots and the length of diseased leaves were investigated, and each data came from the average value of multiple leaves.

4. Analysis of resistance spectrum of genetically transformed plants to bacterial blight

The T1 generation lineages of three genetically transformed plants (OsTFIIAγ5-RNAi1, OsTFIIAγ5-RNAi3 and OsTFIIAγ5-RNAi7) with enhanced resistance in the TO generation were analyzed. Positive plants were detected with specific PCR primers pMCG-f (5'-GGCTCACCAAAACCTTAAACAA-3') and pMCG-r (5'-CTGAGCTACACATGCTCAGGTT-3') on the genetic transformation vector pDS1301. At the booting stage, the field was inoculated with the physiological races PXO99, PXO61, PXO71, PXO79, PXO86, PXO112, PXO341 and PXO347, the physiological races of the Japanese bacterial blight T7174, and the Korean bacterial bacterial races KACC10331. , Chinese bacterial blight physiological races Zhe173, FuJ23, KS-1-21 and YN11. The results showed that the resistance of all plants carrying the pDS1301 vector sequence was significantly enhanced compared to the control Zhonghua 11 (Figure 6). It was proved that inhibition of OsTFIIAγ5 gene expression can enhance the broad-spectrum resistance of rice to bacterial blight.

5. Analysis of the resistance of genetically transformed plants to bacterial leaf streak

The T1 generation lineages of three genetically transformed plants (OsTFIIAγ5-RNAi1, OsTFIIAγ5-RNAi3 and OsTFIIAγ5-RNAi7) with enhanced resistance in the TO generation were analyzed. Positive plants were detected with specific PCR primers pMCG-f (5'-GGCTCACCAAAACCTTAAACAA-3') and pMCG-r (5'-CTGAGCTACACATGCTCAGGTT-3') on the genetic transformation vector pDS1301. Physiological races RH3, RS85, RS105, JSB2-24 and HNB8-47 were inoculated by acupuncture at the 4-5 leaf stage and published 14 days after inoculation (Chen et al., 2006). The results showed that the resistance of all plants carrying the pDS1301 vector sequence was significantly enhanced compared to the control Zhonghua 11 (Figure 7). It was proved that inhibition of OsTFIIAγ5 gene expression can also enhance the resistance of rice to bacterial leaf spot.

Example 3: Point mutation of rice OsTFIIAγ5 gene confers its function in broad-spectrum disease resistance

1. Point mutation of rice OsTFIIAγ5 gene confers broad-spectrum resistance to bacterial blight

Rice lineages IRBB5 and IR24 are near-isogenic lines. IR24 carries the OsTFIIAγ5 gene, and the 39th amino acid of the encoded OsTFIIAγ5 protein is valine (V); IRBB5 carries the mutated OsTFIIAγ5 gene, namely the recessive xa5 gene. The recessive xa5 gene encodes a mutated OsTFIIAγ5 protein (OsTFIIAγ5 ^V39E or xa5) whose amino acid at position 39 is mutated from valine to glutamic acid (E). At the booting stage, the field was inoculated with the physiological races PXO99, PXO339, PXO61, PXO71, PXO79, PXO86, PXO112, PXO341 and PXO347, the physiological races T7174 of bacterial blight in Japan, and the physiological races of bacterial blight in Korea. Species KACC10331, Chinese bacterial blight physiological races Zhe173, FuJ23, KS-1-21 and YN11. The results showed that the resistance of IRBB5 to these bacterial races was significantly enhanced (Fig. 8). It was proved that the mutant OsTFIIAγ5 gene can enhance the resistance of rice to bacterial blight.

2. The point mutation of rice OsTFIIAγ5 gene confers broad-spectrum resistance to bacterial leaf stripe

IRBB5 and IR24 were inoculated with bacterial leaf spot. Physiological races RH3, RS85, RS105, JSB2-24 and HNB8-47 of Chinese bacterial leaf spot were inoculated by acupuncture at the 4-5 leaf stage, and published 14 days after inoculation. The results showed that compared with IR24, IRBB5 significantly enhanced the resistance to these bacterial races of S. striatum (FIG. 9). It was proved that the mutant OsTFIIAγ5 gene can also enhance the resistance of rice to bacterial leaf spot.

3. Mutation of OsTFIIAγ5 does not affect the yield traits of rice

IRBB5 and IR24 were planted naturally in the field with normal water and fertilizer management, and agronomic traits were investigated. The results showed that the agronomic traits of IR24 carrying the OsTFIIAγ5 gene and IRBB5 carrying the xa5 gene were not statistically different (Table 3).

Table 3 Comparison of agronomic traits between rice varieties IR24 and IRBB5

agronomic traits IR24(OsTFIIAγ5) IRBB5(xa5) Heading period (days) 98.00±2.00 97.50±3.50 Sword leaf length (cm) 41.30±1.23 41.42±1.32 Ears per plant 16.00±1.60 16.40±1.10 Ear length (cm) 24.32±0.83 24.52±0.47 grains per ear 198.50±8.00 199.50±7.00 1000-grain weight (g) 24.80±1.78 24.73±1.27 Yield per plant (g) 32.15±4.30 32.00±2.30 Seed setting rate (%) 90.40±4.30 90.10±1.60 Grain length (mm) 6.34±0.13 6.35±0.16 Grain width (mm) 2.13±0.09 2.12±0.17 Grain thickness (mm) 1.85±0.22 1.83±0.17

Example 4: Inhibition of expression of tomato SlTFIIAγ gene to verify its function in disease resistance

1. Construction of a transient transformation vector for inhibiting expression

The present invention adopts virus induced gene silencing (VIGS) technology to verify the function of the gene by inhibiting the expression of the SlTFIIAγ gene in the tomato variety Ailsa Craig. The main mechanism of this technical approach is as follows: a partial fragment of the target gene is linked with a gene silencing vector induced by tobacco rattle virus, and the vector is introduced into tomato through transient transformation to induce the silencing of the target gene in tomato. VIGS technology has been widely used for the verification of tomato gene function (Liu et al., 2002).

The gene silencing vector induced by tobacco crisp virus used in the present invention is pTRV2 (Liu et al., 2002), and the construction diagram of the transformed expression vector pTRV2-SlTFIIAγ is shown in Figure 10). The cDNA fragment of the gene was transferred into the tomato variety Ailsa Craig to inhibit the expression of the SlTFIIAγ gene in Ailsa Craig. The specific operation steps are as follows:

The cDNA clone and transformation vector pTRV2 of tomato SlTFIIAγ gene were digested with restriction enzymes EcoRI and BamHI, and the restriction enzymes were inactivated in a water bath at 65°C for 10 min after the digestion was complete. The enzyme-digested fragment was electrophoresed on a 0.8% agarose gel and the target band was recovered. The exogenous fragment of the cDNA clone was ligated with the transformation vector pTRV2 using T4-DNA ligase. The ligated vector was electrotransformed into Escherichia coli strain DH10B, and the cloned plasmid obtained after electrotransformation was screened by EcoRI and BamHI to verify the positive clone, and named the positive clone as pTRV2-SlTFIIAγ (Figure 10).

2. Transient Transformation and Analysis of Genetically Transformed Plants

pTRV2-SlTFIIAγ was introduced into the tomato variety Ailsa Craig (but not limited to this variety) using the transient transformation method of tobacco rattle virus-induced gene silencing (Liu et al., 2002), while pTRV2-GFP (green fluorescent protein gene) was introduced into tomato Variety Ailsa Craig (but not limited to this variety) served as a control (Liu et al., 2002). After 30 days, all transiently transformed plants were inoculated with tomato bacterial scab (Xanthonmonas campestris pv.vesicatoria) 23-1 (this strain and the following strain tested for pepper spot disease are the same strain, which can harm pepper and tomato at the same time, but cause Disease names vary; Sun et al., 1999). Compared with the transiently transformed plants carrying pTRV2-GFP, the resistance of the transiently transformed plants carrying pTRV2-SlTFIIAγ was significantly enhanced (P<0.01) (see panel A in Figure 11).

In order to further verify whether the enhanced disease resistance of the transiently transformed plants carrying pTRV2-SlTFIIAγ is related to the expression level of SlTFIIAγ gene, the present invention adopts the above real-time quantitative RT-PCR method to detect the expression level of SlTFIIAγ gene in the transiently transformed plants. The test results showed that the expression level of the SlTFIIAγ gene in the transiently transformed plants with enhanced disease resistance was significantly reduced compared with the control (P<0.01B) (see panel B in Figure 11 ). Three days after simultaneous inoculation of tomato bacterial scab, the number of bacteria in the transiently transformed plants with enhanced disease resistance was also significantly reduced compared to the control (P<0.01) (see panel C in Figure 11). The results indicated that the encoded product of the tomato SlTFIIAγ gene played a role as a negative regulator in the anti-bacterial scab response of tomato. Inhibiting the expression of SlTFIIAγ gene in tomato can enhance the resistance of tomato to bacterial scab.

Example 5: Inhibit the expression of pepper CaTFIAγ gene to verify its function in disease resistance

1. Construction of a transient transformation vector for inhibiting expression

The present invention adopts virus-induced gene silencing interference technology to verify the function of the gene by inhibiting the expression of the CaTFIIAγ gene in the pepper variety Hua 50 (not limited to this variety). The main mechanism of this technical approach is as follows: a partial fragment of the target gene is linked to a gene silencing vector induced by tobacco rattle virus, and the vector is introduced into capsicum by transient transformation to induce the silencing of the target gene in capsicum. VIGS technology has been widely used for the validation of pepper gene function (Chung et al., 2004).

The gene silencing vector induced by tobacco crisp virus used in the present invention is pTRV2 (Liu et al., 2002), the modified expression vector is pTRV2-SlTFIIAγ (see Figure 12), and the pepper CaTFIIAγ gene is transformed by the transient transformation method mediated by Agrobacterium. The cDNA fragment was transferred into the pepper variety Hua 50 and inhibited the expression of CaTFIIAγ gene in Hua 50. The specific operation steps are as follows:

The cDNA cloning and transformation vector pTRV2 of pepper CaTFIIAγ gene was digested with restriction enzymes EcoRI and BamHI, and the restriction enzymes were inactivated in a water bath at 65°C for 10 min after the digestion was complete. The enzyme-digested fragment was electrophoresed on a 0.8% agarose gel and the target band was recovered. The exogenous fragment of the cDNA clone was ligated with the transformation vector pTRV2 using T4-DNA ligase. The ligated vector was electro-transformed into E. coli strain DH10B, and the cloned plasmid obtained after electro-transformation was screened by EcoRI and BamHI to verify the positive clone, and named the positive clone as pTRV2-CaTFIIAγ (Figure 12).

2. Transient Transformation and Analysis of Genetically Transformed Plants

pTRV2-CaTFIAγ was introduced into capsicum cultivar Hua 50 using the transient transformation method of tobacco rattle virus-induced gene silencing (Chung et al., 2004), while pTRV2-GFP was introduced into capsicum cultivar Hua 50 as a control (Chung et al., 2004). Thirty days later, all transiently transformed plants were inoculated with pepper spot (Xanthonmonas campestrispv. vesicatoria) 23-1 (see Sun et al., 1999). The resistance of the transiently transformed plants carrying pTRV2-CaTFIAγ was significantly enhanced (P<0.01) compared to the transiently transformed plants carrying pTRV2-GFP (panel A in Figure 13).

To further verify whether the enhanced disease resistance of the transiently transformed plants carrying pTRV2-CaTFIIAγ is related to the expression of the CaTFIIAγ gene, the present invention uses the above real-time quantitative RT-PCR method to detect the expression of the CaTFIIAγ gene in the transiently transformed plants. The test results showed that the expression of the CaTFIAγ gene in the transiently transformed plants with enhanced disease resistance was significantly reduced compared with the control (P<0.01) (see panel B in Figure 13 ). Three days after simultaneous inoculation with pepper spot, the number of bacteria in the transiently transformed plants with enhanced disease resistance was also significantly reduced (P<0.01) compared to the control (see panel C in Figure 13). The results indicated that the encoded product of pepper CaTFIAγ gene played a role as a negative regulator in pepper resistance to pepper spot disease. Inhibiting the expression of CaTFIAγ gene in pepper can enhance the resistance of pepper to pepper spot disease.

Through the functional verification of rice OsTFIIAγ5 gene, tomato SlTFIIAγ gene and pepper CaTFIIAγ gene in plant-pathogen interaction, and the analysis of examples, it is found that inhibiting the expression of OsTFIIAγ5 gene in rice can significantly enhance the resistance of rice to bacterial blight and bacterial blight. Broad-spectrum resistance of various physiological races of bacterial leaf spot; inhibition of SlTFIIAγ gene expression in tomato can enhance tomato resistance to bacterial scab; inhibition of CaTFIIAγ gene expression in pepper can enhance pepper's resistance to pepper spot disease resistance. At the same time, we found that mutant rice OsTFIIAγ5 gene, the recessive xa5 gene, can enhance the broad-spectrum resistance of rice to bacterial blight and bacterial leaf spot races. Therefore, it is possible to search for rice varieties carrying recessive xa5 gene in rice germplasm resources as breeding resources for rice resistance to bacterial blight and bacterial leaf spot. At the same time, the 39th amino acid of OsTFIIAγ5 gene in cultivated rice varieties can also be edited and mutated from valine to glutamic acid by genome DNA editing technology, so as to give rice a broad spectrum of bacterial blight and bacterial leaf spot. resistance.

references

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms. Nucl. Acids Res. 25:3389-3402.

Bart, R., Cohn, M., Kassen, A., McCallum, E.J., Shybut, M., Petriello, A., Krasileva, K., Dahlbeck, D., Medina, C., Alicai, T., et al al.(2012) High-throughputgenomic sequencing of cassava bacterial blight strains identifies conserved effectsors to target for durable resistance.Proc.Natl.Acad.Sci.USA 109:E1972-1979.

Boch J, Bonas U, Lahaye T (2014) TAL effector-pathogen strategies and plant resistance engineering. New Phytologist 204:823-832.

Cao Y, Ding X, Cai M, Zhao J, Lin Y, Li X, Xu C, Wang S(2007) The expression pattern of a rice disease resistance gene Xa3/Xa26is differentially regulated by the genetic backgrounds and developmental stages that influence its function.Genetics 177:523-533.

Chen C, Zheng W, Huang X, Zhang D, Lin X (2006) Major QTL conferringresistance to rice bacterial leaf streak.Agri.Sci.China 5:216-220.

Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, Bennetzen JL, Zhang Q, Wang S(2006) Promoter mutations of an essential gene for pollen developmentresult in disease resistance in rice.Gene.Dev.20:1250-1255.

Chung E, Seong E, Kim YC, Chung EJ, Oh SK, Lee S, Park JM, Joung YH, Choi D (2004) A method of high frequency virus-induced gene silencing in chile pepper (Capsicum annuum L.cv.Bukang ).Mol Cells.17:377-380.

Iyer AS, McCouch SR (2004) The rice bacterial blight resistance gene xa5 encodes a novel form of disease resistance. Mol Plant-Microbe Interact. 17:1348-1354.

Jiang GH, Xia ZH, Zhou YL, Wan J, Li DY, Chen RS, Zhai WX, Zhu LH (2006) Testifying the rice bacterial blight resistance gene xa5 by geneticcomplementation and further analyzing xa5(Xa5) in comparison with its homologTFIIAg1.Mol Genet Genomics. 275:354-366.

Li YF, Le Gourierrec J, Torki M, Kim YJ, Guerineau F, Zhou DX (1999) Characterization and functional analysis of Arabidopsis TFIIA reveal that theevolutionarily unconserved region of the large subunit has a transcriptionactivation domain. Plant Mol. Biol. 39:515 -525.

Lin Y, Zhang Q (2005) Optimising the tissue culture conditions for highefficiency transformation of indica rice. Plant Cell Rep. 23:540-547.

Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing intomato. Plant J. 31:777-786.

F.J.,Potnis,N.,Jones,J.B.,Van Sluys,M.A.,Bogdanove,A.J.,and Dow,J.M.(2011)Pathogenomics of Xanthomonas:understanding bacterium-plant interactions.Nat.Rev.Microbiol.9:344-355.Ryan, RP,

FJ, Potnis, N., Jones, JB, Van Sluys, MA, Bogdanove, AJ, and Dow, JM (2011) Pathogenomics of Xanthomonas: understanding bacterium-plant interactions. Nat. Rev. Microbiol. 9:344-355.

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls forgene activation. Curr. Opin. Microbiol. 14:47-53.

P.,Jordan,T.,and Lahaye,T.(2006)Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effectorproteins.J.Plant Physiol.163:256-272.Schornack, S., Meyer, A.,

P., Jordan, T., and Lahaye, T. (2006) Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effectorproteins. J. Plant Physiol. 163:256-272.

Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression-Total silencing by intron-spliced hairpin RNAs. Nature 407:319-320.

Sun F, Du Z, Jiao Z, Zhao T, Cheng B (1999) Pathogen and raceidentification of bacterial spot of pepper and tomato. Acta Phytopathologica Sinica 29:265-269.

Sun X,Cao Y,Yang Z,Xu C,Li X,Wang S,Zhang Q(2004)Xa26,a geneconferring resistance to Xanthomonas oryzae pv.Oryzae in rice,encoding a LRRreceptor kinase-like protein.Plant J.37: 517-527.

Yuan M, Chu Z, Li X, Xu C, Wang S (2010) The bacterial pathogen Xanthomonasoryzae overcomes rice defenses by regulating host copper redistribution. Plant Cell. 22:3164-3176.

Yudkovsky, N., Ranish, J.A., and Hahn, S. (2000) A transcriptionreinitiation intermediate that is stabilized by activator. Nature 408:225-229.

Zhou B, Peng KM, Chu ZH, Wang SP, Zhang QF (2002) The defense-responsive genes showing enhanced and repressed expression after pathogen infection inrice (Oryza sativa L.). Sci. China Ser C 45:449-467.

Claims (1)

1. A method for cultivating broad-spectrum resistance of rice to bacterial blight and bacterial streak disease is characterized in that an inhibition expression vector is constructed by inhibiting OsTFIIA gamma 5 gene shown as SEQ ID NO. 4, and the expression level of the OsTFIIA gamma 5 gene is reduced by transgenosis to enhance the broad-spectrum resistance of the rice to the bacterial blight and bacterial streak disease; or through mutating protein of OsTFIIA gamma 5 gene, the 39 th amino acid shown in SEQ ID NO. 5 is mutated from valine to glutamic acid, so as to enhance the broad-spectrum resistance of rice to bacterial blight and bacterial streak.

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