CN116042648B - Application of OsGELP77 gene in improving disease resistance of rice - Google Patents
Application of OsGELP77 gene in improving disease resistance of rice Download PDFInfo
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Abstract
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsGELP77 gene in improving disease resistance of rice. The OsGELP77 gene is cloned from rice, and the nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1. Through research on biological functions of the OsGELP77 gene, the excessive expression of the OsGELP77 gene in rice can lead the rice to show disease resistance enhancement capability on a plurality of different pathogenic micro-species of bacterial leaf blight bacteria, bacterial leaf spot bacteria and rice blast bacteria, and the agronomic characters are not obviously changed. The knockout of the OsGELP77 gene in rice can lead the rice to show reduced disease resistance to a plurality of different pathogenic micro-species of bacterial leaf blight bacteria, bacterial leaf spot bacteria and rice blast bacteria, and the agronomic characters of the rice are worsened.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsGELP77 gene in improving rice disease resistance, wherein the gene can be applied to cultivation and enhancement of rice variety improvement with broad-spectrum bacterial leaf blight resistance, bacterial leaf streak resistance and rice blast resistance.
Background
The rice is subjected to various disease damages in production, wherein bacterial leaf blight and bacterial leaf streak, fungal disease rice blast are the most serious diseases affecting the rice production, and particularly have obvious influence on the safe production of rice in China and even the world. According to researches, the rice blast can cause 10-35% of yield reduction of the rice, bacterial leaf blight can cause 20-30% of yield reduction of the rice, and bacterial leaf streak can cause about 20% of yield reduction of the rice. In recent decades, with climate change, large-area popularization of high-quality hybrid rice and nitrogen hypertrophy application, the fungi and bacterial diseases occur in large areas in various rice production areas in China, and the fungi and bacterial diseases tend to increase year by year. Therefore, the disease resistance genes of the rice are developed and effectively utilized, and the rice variety is improved, so that the rice is urgent to improve the disease resistance of the rice to different diseases.
The disease-resistant response of plants is a complex process of polygenic control. Genes involved in plant disease resistance are divided into two classes: a main effect disease-resistant gene and a disease-resistant QTL gene. However, the resources of the major disease-resistant gene are limited, and the major disease-resistant gene is only resistant to one disease or only resistant to one or a few pathogenic micro-species of a certain disease, and the disease-resistant range is limited. At present, only major disease-resistant genes for resisting rice blast and bacterial leaf blight are identified and cloned, and no major disease-resistant genes for resisting bacterial leaf streak are found or identified. In addition to the major disease resistance genes, some disease resistance QTL genes have been identified, a few of which have been cloned, and the proteins they encode are involved in the synthesis of disease resistance signaling molecules, signaling, defense responses, such as this metabolite, etc. in plants. However, in the process of effectively utilizing the disease-resistant QTL genes, part of disease-resistant QTL genes are found to have negative effects on important agronomic traits of rice, so that the breeding application of the disease-resistant QTL genes is limited to a certain extent.
According to the research, the OsGELP77 gene in rice is induced to express by bacterial leaf blight, bacterial leaf spot and rice blast, and the overexpression of the OsGELP77 gene can enhance the broad-spectrum disease resistance of the rice to bacterial leaf blight, bacterial leaf spot and rice blast, and has no obvious influence on important agronomic characters of the rice. And the knockout of the OsGELP77 gene can reduce the broad-spectrum disease resistance of rice to bacterial leaf blight, bacterial leaf streak and rice blast. The invention has important significance on the improvement of rice with important grain crops on bacterial leaf blight resistance, bacterial leaf streak resistance and rice blast resistance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and researches on biological functions of the OsGELP77 gene find that the OsGELP77 gene has important regulation and control functions on bacterial leaf blight resistance, bacterial leaf blight resistance and rice blast resistance of rice, and the broad-spectrum disease resistance of the rice to bacterial leaf blight, bacterial leaf blight and rice blast is influenced by regulating the expression level of the OsGELP77 gene. Therefore, the invention has important significance on the improvement of rice for important grain crops, such as bacterial leaf blight, bacterial leaf streak and rice blast.
The technical scheme of the invention is as follows:
the invention proves that the overexpression of the OsGELP77 gene in rice can lead the rice to show the capability of enhancing the disease resistance to a plurality of different pathogenic micro-species of bacterial leaf blight bacteria, bacterial strip spot bacteria and rice blast bacteria. The broad-spectrum disease resistance of rice to bacterial leaf blight, bacterial leaf streak and rice blast can be reduced by knocking out the OsGELP77 gene. Through systematic research, the applicant finds that the OsGELP77 gene has important regulation and control functions in the aspects of rice bacterial leaf blight resistance, bacterial leaf streak resistance and rice blast resistance.
Biological function verification proves that the rice OsGELP77 gene provided by the invention has the following characteristics:
1. the nucleotide sequence of the OsGELP77 gene is shown in a sequence table SEQ ID NO. 1.
The nucleotide sequence shown in SEQ ID NO. 1 consists of 2289 deoxyribonucleotides of rice OsGELP77 gene and upstream and downstream non-coding sequences thereof. Deoxyribonucleotides from 1 st to 177 th in the sequence shown in SEQ ID NO. 1 are upstream non-coding sequences of the OsGELP77 gene; deoxyribonucleotides 178 to 412 are the first exon sequence of the osgel 77 gene; the 413 th to 545 th deoxyribonucleotide is the first intron sequence of the OsGELP77 gene; the 546 th to 755 th deoxyribonucleotide is the second exon sequence of the OsGELP77 gene; deoxyribonucleotides 756 to 842 are the second intron sequence of the OsGELP77 gene; the deoxyribonucleotides 843 to 1265 are the third exon sequence of the OsGELP77 gene; deoxyribonucleotides 1266 to 1727 are the third intron sequence of the osgel 77 gene; the deoxyribonucleotide at positions 1728 to 1942 is the fourth exon sequence of the OsGELP77 gene; the deoxyribonucleotides 1943 to 2289 are the downstream non-coding sequence of the OsGELP77 gene.
2. The OsGELP77 gene sequence related by the invention can be applied to crops, especially rice disease-resistant breeding, transgenic lines and new varieties.
For more details, reference is made to the description of the embodiments.
Compared with the prior art, the invention has the beneficial effects that:
overexpression of the OsGELP77 gene enhances the broad-spectrum disease resistance of rice to bacterial leaf blight, bacterial leaf streak and rice blast. The knockout of the OsGELP77 gene can reduce the broad-spectrum disease resistance of rice to bacterial leaf blight, bacterial leaf streak and rice blast.
Drawings
Fig. 1: the invention identifies and separates cloned rice OsGELP77 gene and verifies the technical flow chart of OsGELP77 gene function.
Fig. 2: the quantitative reverse transcription-PCR (quantitative reverse transcription-PCR, RT-qPCR) technology is used for detecting the expression mode of the OsGELP77 gene after the rice variety flower 11 is inoculated with different pathogenic bacteria respectively. Reference numerals illustrate: panel A in FIG. 2 shows the expression level of the OsGELP77 gene after the rice is inoculated with bacterial blight PXO99, the expression level of the OsGELP77 gene in each sample is 0 hours relative to the expression level of the rice after the rice is inoculated with bacterial blight 11, and each data is the average (3 replicates). + -standard deviation; panel B in FIG. 2 shows the level of the OsGELP77 gene expressed after inoculating rice with bacterial leaf spot germ GX01, the OsGELP77 gene expressed in each sample was expressed relative to the rice flower 11 inoculated with bacterial leaf spot germ for 0 hour, and each data was the average (3 replicates). + -standard deviation; panel C in FIG. 2 shows the level of the OsGELP77 gene expression after rice is inoculated with Pyricularia oryzae M2, and the OsGELP77 gene expression level in each sample was 0 days relative to that of rice inoculated with Pyricularia oryzae 11 flowers. Each data is the mean (3 replicates) ± standard deviation.
Fig. 3: vector map of the genetic transformation vector pU1301 used in the present invention. Reference numerals illustrate: FIG. 3 shows a vector map of pU1301. Wherein RB and LB represent the right and left border of T-DNA, respectively, GUS represents the beta-glucuronidase gene, hpt represents the hygromycin phosphotransferase gene, PUbi represents the maize ubiquitin gene promoter, TEVL represents the 5' untranslated region of tobacco etch virus, NOS represents the polyadenylation signal of the nopaline synthase gene. The OsGELP77 gene sequence is ligated into the over-expression vector pU1301 under the driving of the constitutive promoter PUbi. The vector is used to transform wild rice (namely the non-transgenic rice variety) so as to over express the OsGELP77 gene in rice.
Fig. 4: and detecting the expression level of the OsGELP77 gene in the rice line with the over-expressed OsGELP77 gene. Reference numerals illustrate: the expression level of the OsGELP77 gene in 8 transgenic individuals of OsGELP77 gene overexpression (OsGELP 77-OE) is compared with the transgenic background wild type.
Fig. 5: vector map of the genetic transformation vector pYLCRISPR/Cas9-MH (KR 029109) used in the present invention. Reference numerals illustrate: FIG. 5 shows a vector map of pYLCRISPR/Cas 9-MH. Wherein: p (P) 35 : HPT represents hygromycin phosphotransferase gene expressed by 35S promoter of cauliflower mosaic virus; p (P) Ubi Represents a maize ubiquitin gene promoter; NLS represents the nuclear localization signal sequence; t (T) nos Represents the Agrobacterium tumefaciens terminator sequence.
Fig. 6: the invention relates to design sites and genotype detection of target sites of OsGELP77 gene knockout rice strains. Reference numerals illustrate: panel A in FIG. 6 shows the design sites for the U3 promoter to direct gRNA and the U6a promoter to direct gRNA; panel B in FIG. 6 shows the number and positions of deoxyribonucleotides deleted from OsGELP77 gene knockout rice lines OsGELP77-1 and OsGELP77-2 compared with the wild type; panel C in FIG. 6 shows that PCR products for OsGELP77 gene knockout rice lines OsGELP77-1 and OsGELP77-2 were significantly smaller than those of wild type rice by agarose gel electrophoresis.
Fig. 7: the OsGELP77 gene over-expression strains OsGELP77-OE6 and OsGELP77-OE10, the knock-out rice strains OsGELP77-1 and OsGELP77-2 are inoculated with different bacterial blight bacteria (PXO 61, PXO71, PXO341, PXO347, from Philippine International Rice research institute) for lesion length. Reference numerals illustrate: the lengths of the lesions of the OsGELP77 gene overexpression strain OsGELP77-OE6 and OsGELP77-OE10 inoculated with different bacterial leaf blight rice are obviously shortened compared with those of wild rice plants; the length of the lesion of the OsGELP77 gene knocked-out rice strains OsGELP77-1 and OsGELP77-2 inoculated with different bacterial leaf blight bacteria is obviously longer than that of a wild rice plant. The result shows that the rice strain with the over-expressed OsGELP77 gene shows a phenotype with enhanced broad-spectrum disease resistance to different bacterial blight bacteria, and the rice strain with the knocked-out OsGELP77 gene shows a phenotype with reduced disease resistance to different bacterial blight bacteria.
Fig. 8: the OsGELP77 gene over-expression strains OsGELP77-OE6 and OsGELP77-OE10, the knock-out rice strains OsGELP77-1 and OsGELP77-2 are inoculated with different bacterial leaf streaks (RH 3, GX01, HNB8-47 and RS105, and the professor Chen Gongyou of Shanghai university of transportation). Reference numerals illustrate: compared with a wild plant, the length of the disease spots of the OsGELP77 gene over-expression strain OsGELP77-OE6 and OsGELP77-OE10 inoculated with different bacterial strip spot pathogens is obviously shortened; the length of the disease spots of the OsGELP77 gene knockout rice strains OsGELP77-1 and OsGELP77-2 inoculated with different bacterial strip spot pathogens is obviously longer than that of wild plants. The result shows that the rice strain with the over-expressed OsGELP77 gene shows a phenotype with enhanced broad-spectrum disease resistance to different bacterial leaf scald germs, and the rice strain with the knocked-out OsGELP77 gene shows a phenotype with reduced disease resistance to different bacterial leaf scald germs.
Fig. 9: the OsGELP77 gene over-expression strains OsGELP77-OE6 and OsGELP77-OE10, and the knocked-out rice strains OsGELP77-1 and OsGELP77-2 are inoculated with the rice blast fungus M2. Reference numerals illustrate: the lengths of the lesions of the OsGELP77 gene overexpression strain OsGELP77-OE6 and OsGELP77-OE10 inoculated with the Magnaporthe grisea are obviously shortened compared with those of wild plants; the length of the lesion after the OsGELP77 gene knockout rice strains OsGELP77-1 and OsGELP77-2 are inoculated with the rice blast bacteria is obviously longer than that of the wild type plants. The result shows that the rice strain with the over-expressed OsGELP77 gene shows a phenotype with enhanced broad-spectrum disease resistance to the rice blast bacteria, and the rice strain with the knocked-out OsGELP77 gene shows a phenotype with reduced disease resistance to the rice blast bacteria.
Detailed Description
Description of the sequence Listing:
the nucleotide sequence of the OsGELP77 gene is shown in the sequence table SEQ ID NO. 1. The sequence length is 2289bp.
The invention is further illustrated below in connection with specific examples. FIG. 1 depicts a flow for identifying and isolating cloned OsGELP77 gene and validating the function of OsGELP77 gene. It is noted that these examples are only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the claims in any way.
The methods used in the following examples are conventional methods unless otherwise specified, and reference may be made to the following steps: molecular Cloning A laboratory; ry Manual (Sambrook, j., russell, david w., molecular Cloning: A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor) or related products. The reagents or instruments used are not specific to manufacturers and are conventional products purchased through the market.
Example 1: analysis of OsGELP77 Gene expression Pattern after treatment of Rice with different pathogenic bacteria
To verify whether the oscelep 77 gene is involved in regulating disease resistance of rice. The applicant respectively inoculates bacterial leaf blight bacteria PXO99, bacterial leaf spot bacteria GX01 and rice blast bacteria M2 on the leaves of a booting rice variety 'Zhonghua 11' (the variety is a mode variety for public and scientific research from the national academy of agricultural sciences of China), and analyzes the expression mode of OsGELP77 genes in response to pathogenic bacteria by adopting RT-qPCR technology, wherein the primers are OsGELP77RTF (5'-CATGTACGCCGACTTCTACTC-3') and OsGELP77RTR (5'-GCCCTCAAATTCGTGCTAAAC-3'). The result shows that the OsGELP77 gene can be induced to express by bacterial leaf blight bacteria, bacterial leaf streak bacteria and rice blast bacteria. This result suggests that: the OsGELP77 gene may be involved in regulating the resistance reaction of rice to bacterial leaf blight, bacterial leaf streak and rice blast (see FIG. 2 for results).
Example 2: obtaining rice material of over-expression OsGELP77 gene
(1) Construction of overexpression OsGELP77 Gene expression vector
This example is a general description of pU1301-OsGELP77 vector construction.
First, the primers OsGELP77F (5'-ATTTACGAACGATAGCCGGTACCGGCATTGCCTCATCCAT-3') and OsGELP77R (5'-TGCAGGTCGACTCTAGAGGATCCGGGCATGATCGGCGGGT-3') are designed by taking the DNA of Zhonghua 11 rice as a template, and the gene sequence of OsGELP77 is amplified by using high-fidelity DNA polymerase PCR. The PCR product was recovered by electrophoresis and ligated to the cloning vector pGEM-T-EASY vector, E.coli DH 5. Alpha. Was transformed, and the monoclonal was obtained by plating. The correctly sequenced clone was selected to extract the plasmid, and the fragment of the OsGELP77 gene was recovered by cleavage with KpnI and BamHI. At the same time, vector pU1301 (map see FIG. 3) was digested overnight with KpnI and BamHI andand (5) recycling. The recovered osgel 77 gene fragment was combined with the vector fragment in a molar ratio of about 3:1 (T4 Ligase) Overnight. The following day, E.coli DH 5. Alpha. Was transformed with the ligation product and cultured overnight at 37℃to obtain a monoclonal. Then, the obtained monoclonal is selected for culturing, meanwhile, PCR verification is carried out on the monoclonal, plasmids are extracted from the clones with correct verification, and further sequencing verification is carried out, so that the pU1301-OsGELP77 vector for plant transformation is obtained.
(2) Obtaining and identifying rice strain of over-expressed OsGELP77 gene
The applicant transferred pU1301-OsGELP77 vector into flower 11 of rice variety by agrobacterium-mediated genetic transformation method to obtain 8 independent transgenic lines. The expression level of the OsGELP77 gene in these lines was analyzed by RT-qPCR technique, wherein the primers used were OsGELP77RTF (5'-CATGTACGCCGACTTCTACTC-3') and OsGELP77RTR (5'-GCCCTCAAATTCGTGCTAAAC-3'). The results showed that the expression level of the osgel 77 gene in these 8 independent transgenic lines was significantly higher than that in the wild type control (see fig. 4). Randomly selecting OsGELP77 gene over-expression rice strain OsGELP77-OE6 and OsGELP77-OE10 to carry out subsequent experiments.
Example 3: obtaining OsGELP77 gene knock-out rice material
(1) Construction of OsGELP77 Gene knockout vector
This example is a general description of the construction of the pYLCRISPR/Cas9-MH-OsGELP77 vector. Primers OsGELP 77U 3F (5'-GGCAGGCTGGGTGAGCGTCAGGCGTTTTAGAGCTAG-3') and OsGELP 77U 3R (5'-GCCTGACGCTCACCCAGCCTGCCACGGATCATCTGC-3') are designed, which can specifically match the 320 th to 339 th deoxyribonucleotides in the sequence shown in SEQ ID NO. 1, osGELP 77U 6aF (5'-GCCGGTTGCGTCCGCCATGTTCGGTTTTAGAGCTAG-3') and OsGELP 77U 6aR (5'-CGAACATGGCGGACGCAACCGGCAGCCAAGCCAGCAC-3'), and which can specifically match the 207 th to 226 th deoxyribonucleotides in the sequence shown in SEQ ID NO. 1, as shown in FIG. 6A. 2 rounds of nested PCR were performed: performing 2 reactions in the first round of PCR, namely performing PCR reactions by taking a U-F (5'-CTCCGTTTTACCTGTGGAATCG-3')/OsGELP 77U 3R and an OsGELP 77U 3F/gRNA-R (5'-CGGAGGAAAATTCCATCCAC-3') as DNA templates and taking a pYLgRNA-OsU vector (KR 029104) as a DNA template to obtain products (1) and (2), and performing PCR reactions by taking a U-F/OsGELP 77U 6aR and an OsGELP 77U 6aF/gRNA-R as templates and taking a pYLgRNA-OsU6a vector (KR 029105) as templates to obtain products (3) and (4); the second round is overlap PCR, i.e., PCR with primers Up-T1 (5'-ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTGGAATCGGCAGCAAAGG-3') and gR-T1 (5'-CAGGGAGCGGATAACAATTTCACACAGGCACATCCACTCCAAGCTCTTG-3') using (1) and (2) as templates to obtain product (5), and PCR with primers Up-T2 (5'-GTGCCTGTGTGAAATTGTTATCCGCTCCCTGGAATCGGCAGCAAAGG-3') and gR-T2 (5'-CCACGCATACGATTTAGGTGACACTATAGCGCATCCACTCCAAGCTCTTG-3') using (3) and (4) as templates to obtain product (6). Cleavage of the pYLCRISPR/Cas9-MH vector with BsaI (see FIG. 5); the PCR products (5), (6) and BsaI digested pYLCRISPR/Cas9-MH vector were combined according to 2:2:1 volume was mixed, reacted with pEASY-Uni Seamless Cloning and Assembly Kit (Beijing full gold Biotechnology Co., ltd.) at 50℃for 15 minutes, and E.coli DH 5. Alpha. Was transformed with the reaction product and cultured overnight at 37℃to obtain a monoclonal antibody. The monoclonal is selected for culturing, meanwhile, PCR verification is carried out on the monoclonal, plasmids are extracted from the clones with correct verification, and further sequencing verification is carried out. Thereby obtaining a plant genetic transformation vector (pYLCRISPR/Cas 9-MH-OsGELP 77).
(2) Obtaining and identifying OsGELP77 gene knockout rice strain
The applicant introduced plant genetic transformation vector pYLCRISPR/Cas9-MH-OsGELP77 into flower 11 of oryza sativa variety by agrobacterium-mediated genetic transformation. Multiple independent transgenic families are obtained, family 1 (named OsGELP 77-1) and family 2 (named OsGELP 77-2) are selected for subsequent experiments, and primers OsGELP77 CgF (5'-GGTGAGTAAGTTGAAGGTGGTC-3') and OsGELP77CgR (5'-AGGGAGTTATGGCGGGAG-3') are used for PCR amplification of the wild type and OsGELP77 gene knockout rice strains, namely family 1 and family 2, and the gene knockout condition is identified. Line 1 of the OsGELP77 gene knockout rice line lacks 99 deoxyribonucleotides between two design target sites, and line 2 lacks 100 deoxyribonucleotides between two design target sites. The results are shown in FIG. 6, panel B. Agarose gel electrophoresis using PCR products showed that large fragment deoxyribonucleotide deletions were present in both line 1 and line 2 of OsGELP77 gene knockout rice line at the genomic level (see panel C in FIG. 6 for results).
Example 4: disease resistance analysis of OsGELP77 gene over-expression rice strain and knockout rice strain
(1) OsGELP77 gene over-expression rice strain and bacterial leaf blight resistance phenotype analysis of knockout rice strain
In China, hubei, wuhan summer fields carry out bacterial blight inoculation tests on OsGELP77 gene over-expressed rice strains OsGELP77-OE6 and OsGELP77-OE10, knocked rice strains OsGELP77-1 and OsGELP77-2 and wild type controls. The results show that compared with a wild type (non-transgene, the same applies hereinafter), the disease length of the OsGELP77 gene over-expression rice strain OsGELP77-OE6 and OsGELP77-OE10 is obviously shorter than that of a wild type control (p < 0.01) when the OsGELP77 gene over-expression rice strain of the invention is inoculated with different bacterial leaf blight pathogenic minispecies (such as PXO61, PXO71, PXO341 and PXO347 from Philippine International paddy institute). Compared with a wild type (non-transgene, the same applies hereinafter), the OsGELP77 gene knockout rice strain disclosed by the invention has the advantages that the onset lengths of the OsGELP77 gene knockout rice strain OsGELP77-1 and OsGELP77-2 are obviously longer than those of a wild type control (p < 0.01) when different bacterial blight pathogenic minispecies (PXO 61, PXO71, PXO341 and PXO 347) are inoculated in the booting stage. See fig. 7. The result shows that the rice strain with the over-expressed OsGELP77 gene can enhance the broad-spectrum disease resistance of rice to a plurality of pathogenic micro-species of bacterial leaf blight, and the rice strain with the over-expressed OsGELP77 gene can reduce the disease resistance of rice to a plurality of pathogenic micro-species of bacterial leaf blight.
(2) OsGELP77 gene over-expression rice strain and bacterial leaf spot resistance phenotype analysis of knockout rice strain
In China, hubei, wuhan summer fields carry out bacterial leaf spot germ inoculation experiments on OsGELP77 gene over-expressed rice strains OsGELP77-OE6 and OsGELP77-OE10 and knock-out rice strains OsGELP77-1 and OsGELP77-2 and wild controls. The result shows that the OsGELP77 gene over-expression rice strain of the invention is inoculated with different pathogenic species of bacterial leaf scald germ (RH 3, GX01, HNB8-47, RS105 and strains taught by Shanghai university of transportation Chen Gongyou) in the booting stage, and compared with a wild type (non-transgene, the disease incidence length of the OsGELP77 gene over-expression rice strain OsGELP77-OE6 and OsGELP77-OE10 is obviously shorter than that of a wild type control (p < 0.01). Compared with wild type (non-transgene, the same applies hereinafter), the OsGELP77 gene knockout rice strain of the invention is inoculated with different pathogenic species (RH 3, GX01, HNB8-47, RS 105) of bacterial leaf spot bacteria during booting period, and compared with wild type (non-transgene, the same applies hereinafter), the disease length of the OsGELP77 gene knockout rice strain OsGELP77-1 and OsGELP77-2 is obviously longer than that of wild type control (p < 0.01). See fig. 8. The result shows that the rice strain with the over-expressed OsGELP77 gene can enhance the broad-spectrum disease resistance of rice to a plurality of pathogenic micro-species of bacterial leaf scald germs, and the rice strain with the over-expressed OsGELP77 gene can reduce the disease resistance of rice to a plurality of pathogenic micro-species of bacterial leaf scald germs.
(3) Rice blast resistance phenotype analysis of OsGELP77 gene over-expression rice line and knockout rice line
In China, hubei, wuhan summer fields are used for carrying out rice blast fungus inoculation experiments on OsGELP77 gene over-expressed rice lines OsGELP77-OE6 and OsGELP77-OE10 and knock-out rice lines OsGELP77-1 and OsGELP77-2 and wild type controls. The result shows that compared with a wild type (non-transgene, the same applies hereinafter), the disease length of the OsGELP77 gene over-expressed rice strain OsGELP77-OE6 and OsGELP77-OE10 is obviously shorter than that of a wild type control (p < 0.01) when the rice strain with the OsGELP77 gene over-expressed rice gene is inoculated with rice blast fungus pathogenic micro-seed M2 in the booting stage. Compared with a wild type (non-transgene, the same applies hereinafter), the OsGELP77 gene knockout rice strain is inoculated with rice blast fungus M2 in the booting stage, and the attack length of the OsGELP77 gene knockout rice strain OsGELP77-1 and OsGELP77-2 is obviously longer than that of a wild type control (p < 0.01). See fig. 9. The result shows that the rice plant line with the OsGELP77 gene over-expression can enhance the disease resistance of rice to Pyricularia oryzae, and the rice plant line with the OsGELP77 gene knockout expression can reduce the disease resistance of rice to Pyricularia oryzae.
Example 5: analysis of agronomic traits of OsGELP77 Gene over-expressed Rice Strain and knockout Rice Strain
In China, wild (non-transgenic, the same applies below) and OsGELP77 genes are planted in summer on an internal scientific research base of agricultural university in Wuhan China, hubei, so that rice strains OsGELP77-OE6 and OsGELP77-OE10 are overexpressed, and rice strains OsGELP77-1 and OsGELP77-2 are knocked out. 100 plants are planted in each part of material, and normal water and fertilizer management is performed.
Table 1 agronomic trait investigation and statistical analysis of OsGELP77 Gene over-expressed Rice lines
Table 2 agronomic trait investigation and statistical analysis of OsGELP77 Gene knockout Rice lines
Harvesting rice after autumn maturity, and investigating agronomic characters including plant height, tillering number, spike length, sword leaf width, grain length, grain width, thousand grain weight, fruiting rate and single plant yield. The results show that there is no significant difference in the above agronomic traits of OsGELP77 gene over-expressed rice lines OsGELP77-OE6 and OsGELP77-OE10 compared to the non-transgenic wild type control (Table 1). The OsGELP77 gene knockout rice lines OsGELP77-1 and OsGELP77-2 have no significant difference in plant height, leaf length, leaf width, grain width compared to the non-transgenic wild type control, while tillering number, ear length, grain length, thousand grain weight, seed setting rate, individual plant yield were significantly reduced compared to the non-transgenic wild type control (p < 0.01) (see table 2).
Claims (1)
1. Overexpression ofOsGELP77The application of the gene in cultivating rice varieties with broad-spectrum bacterial leaf blight resistance, bacterial leaf streak resistance and rice blast resistance is characterized in thatOsGELP77The nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1.
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| CN112195184A (en) * | 2020-10-04 | 2021-01-08 | 华中农业大学 | Application of OsMAPK6 gene in improving disease resistance of rice |
| CN112266922A (en) * | 2020-10-02 | 2021-01-26 | 华中农业大学 | Application of OsMAPKK4 gene in improvement of disease resistance of rice |
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| CN112266922A (en) * | 2020-10-02 | 2021-01-26 | 华中农业大学 | Application of OsMAPKK4 gene in improvement of disease resistance of rice |
| CN112195184A (en) * | 2020-10-04 | 2021-01-08 | 华中农业大学 | Application of OsMAPK6 gene in improving disease resistance of rice |
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