Application of rice OsLBD gene in improving resistance of rice to bacterial blight

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a rice OsLBD gene to improvement of bacterial blight resistance of rice, and the nucleotide sequence of the OsLBD gene is shown as SEQ ID No.1. According to the invention, a regulation mechanism of rice disease resistance is disclosed from a molecular level, a new related gene for regulating disease resistance is found, and the gene is applied to inhibition of rice bacterial leaf blight, so that a foundation is laid for cultivation of rice bacterial leaf blight resistant varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of a rice OsLBD gene in improving the resistance of rice to bacterial blight. Background Art

[0002] Rice, as an annual herbaceous plant of the genus Oryza in the family Poaceae, is a major food crop and is closely related to people's lives. In recent years, diseases on rice have seriously threatened rice production, mainly including rice blast, false smut, and bacterial blight of rice.

[0003] Bacterial blight of rice is the world's largest bacterial disease of rice, mainly caused by the infection of Xanthomonas oryzae pv. Oryzae. It is one of the main diseases in rice production. Xanthomonas oryzae pv. Oryzae is abbreviated as Xoo. Its occurrence and epidemic lead to a decrease in the seed setting rate and 1000-grain weight of rice. With the change of climate conditions and planting methods, the occurrence frequency and damage degree of bacterial blight of rice are increasing year by year, posing a major challenge to the yield and quality of rice.

[0004] The impacts of these diseases on rice yield and quality are the most serious. Although the use of pesticides has reduced the impact of diseases, there are problems such as pesticide residues and negative impacts on the natural environment. Therefore, there is an urgent need to provide a new strategy to deal with bacterial blight of rice. Summary of the Invention

[0005] To reduce the damage caused by bacterial blight of rice, the present invention provides the application of a rice OsLBD gene in improving the resistance of rice to bacterial blight.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, there is provided the application of a rice OsLBD gene in improving the resistance of rice to bacterial blight, and the nucleotide sequence of the OsLBD gene is as shown in SEQ ID No.1.

[0008] In the second aspect of the present invention, there is provided an overexpression vector, and the overexpression vector contains the nucleotide sequence.

[0009] In the third aspect of the present invention, there is provided a preparation method of the overexpression vector, including the following steps:

[0010] Synthesize a pair of primers, and the sequences of the primers are as shown in SEQ ID No.3 and SEQ ID No.4;

[0011] Extract rice RNA, reverse transcribe it into cDNA, and use the cDNA as a template to perform PCR amplification with the primers to obtain the OsLBD gene;

[0012] The OsLBD gene was ligated to the digested expression vector and transformed to obtain the overexpression vector.

[0013] Preferably, the expression vector includes any one of the pCAMBIA series vectors and the pBI series vectors.

[0014] Preferably, any one of pCAMBIA3301, pCAMBIA1300, and pCAMBIA1305 is included in the pCAMBIA series vectors;

[0015] Any one of pBI121 and pBI101 is included in the pBI series vectors;

[0016] The fourth aspect of the present invention provides an application of the overexpression vector, and the overexpression vector is used to prepare a rice plant overexpressing the OsLBD gene. The preparation process includes the following steps:

[0017] Prepare an Agrobacterium liquid containing the overexpression vector and use the Agrobacterium liquid to infect rice;

[0018] Cultivate the infected rice to obtain a rice plant overexpressing the OsLBD gene.

[0019] Preferably, when infecting rice, the rice variety used for infection is Nipponbare.

[0020] Preferably, use the Agrobacterium liquid to infect the callus of rice.

[0021] The fifth aspect of the present invention provides an application of the overexpression vector, and the overexpression vector is used to improve the resistance of rice to bacterial blight.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The present invention provides an application of a rice OsLBD gene in improving the resistance of rice to bacterial blight. The nucleotide sequence of the OsLBD gene is shown in SEQ ID No.1. The present invention reveals the regulatory mechanism of rice disease resistance at the molecular level, finds a new regulatory gene related to disease resistance, and applies it to inhibit bacterial blight of rice, laying a foundation for cultivating rice varieties resistant to bacterial blight.

[0024] The present invention has found through research that the rice genes OsLBD11 / OsLBD12 play an important role in the process of rice resistance to bacterial blight. The deletion of the OsLBD11 / OsLBD12 gene reduces the basic defense ability of rice against Xanthomonas oryzae pv. oryzae, while the overexpression of the OsLBD11 / OsLBD12 gene in rice can significantly improve the basic defense ability of rice against Xanthomonas oryzae pv. oryzae. Therefore, the rice OsLBD11 / OsLBD12 gene can be used to regulate the resistance of rice to bacterial blight.

[0025] Disease-resistant breeding is currently the most economical and effective method to resist pathogens. Exploring new bacterial blight-resistant genes, resistance resources, and cultivating broad-spectrum disease-resistant varieties is a green and efficient strategy to ensure crop yield and quality, and is of great significance to agricultural production. Brief Description of the Drawings

[0026] Figure 1 It is a diagram showing the identification results of the resistance of rice overexpression lines to bacterial blight. A is a diagram of inoculation in greenhouse pots. From left to right are 3 parallel samples of NIP, 3 parallel samples of OsLBD11 / 12-OE1, and 3 parallel samples of OsLBD11 / 12-OE2; B is a statistical chart of the lesion length of diseased leaves, where *** represents a significant difference with P<0.001.

[0027] Figure 2 It is a diagram showing the expression results of rice defense-related genes in rice overexpression lines. A is the detection result of the relative expression level of the OsPR1b gene after treatment with flg22; B is the detection result of the relative expression level of the OsPR10 gene after treatment with flg22; C is the detection result of the relative expression level of the OsPR1b gene after treatment with chitin; D is the detection result of the relative expression level of the OsPR10 gene after treatment with chitin, where a, b represent a significant difference with P<0.05.

[0028] Figure 3 It is a diagram showing the identification results of the resistance of rice knockout lines to bacterial blight. A is a diagram of inoculation in greenhouse pots. From left to right are 3 parallel samples of ZH11, 3 parallel samples of oslbd11 / 12-22, and 3 parallel samples of oslbd11 / 12-24; B is a statistical chart of the lesion length of diseased leaves, where *** represents a significant difference with P<0.001.

[0029] Figure 4It is a figure showing the expression results of rice defense-related genes in rice knockout lines. A shows the detection results of the relative expression level of the OsPR1b gene after treatment with flg22; B shows the detection results of the relative expression level of the OsPR10 gene after treatment with flg22; C shows the detection results of the relative expression level of the OsPR1b gene after treatment with chitin; D shows the detection results of the relative expression level of the OsPR10 gene after treatment with chitin, where a and b represent significant differences with P < 0.05. Detailed implementation manners

[0030] The present invention will be further described below through specific embodiments, but it does not limit the scope of the present invention. Without departing from the spirit and scope of the present invention, modifications or substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications or substitutions all fall within the protection scope of the present invention.

[0031] The inventive concept of the present invention is as follows:

[0032] Rice bacterial blight is the world's largest bacterial disease of rice, mainly caused by the infection of Xanthomonas oryzae pv. Oryzae. It is one of the main diseases in rice production. Xanthomonas oryzae is abbreviated as Xoo. Its occurrence and prevalence lead to a decrease in the seed setting rate and a reduction in the 1000-grain weight of rice. With the changes in climate conditions and planting methods, the occurrence frequency and damage degree of rice bacterial blight have increased year by year, posing a major challenge to the yield and quality of rice.

[0033] These diseases have the most serious impact on the yield and quality of rice. The use of pesticides has reduced the impact of diseases, but the use of pesticides has pesticide residues and negative impacts on the natural environment. Therefore, there is an urgent need to provide a new strategy for dealing with rice bacterial blight.

[0034] Based on this, the present invention provides an application of a rice OsLBD gene in improving the resistance of rice to bacterial blight. The present invention reveals the regulatory mechanism of rice disease resistance at the molecular level, finds a new regulatory gene related to disease resistance, and applies it to inhibit rice bacterial blight, laying a foundation for cultivating rice varieties resistant to bacterial blight. The present invention has found through research that the rice genes OsLBD11 / OsLBD12 play an important role in the process of rice resistance to bacterial blight. Rice overexpressing the OsLBD11 / OsLBD12 gene can significantly improve the basic defense ability of rice against Xanthomonas oryzae pv. Oryzae. Therefore, the rice OsLBD11 / OsLBD12 gene can be used to regulate the resistance of rice to bacterial blight.

[0035] To enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention will be further described below in conjunction with specific embodiments. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0036] Example 1

[0037] The application of the rice OsLBD gene in improving the resistance of rice to bacterial blight is as follows:

[0038] 1. Obtaining of overexpression mutants of the rice OsLBD gene.

[0039] The CDS sequence of the rice OsLBD gene is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2.

[0040] The OsLBD gene described in the present invention includes OsLBD11 and OsLBD12. The gene number of OsLBD11 is LOC_Os11g01550; the gene number of OsLBD12 is LOC_Os12g01550; OsLBD11 and OsLBD12 are located on different chromosomes, but their CDS sequences are exactly the same. Therefore, based on the sequence shown in SEQ ID No.1, the present invention constructed plants overexpressing OsLBD11 or OsLBD12.

[0041] The present invention overexpresses the sequence shown in SEQ ID No.1 using the pCAMBIAI1305 vector to obtain an overexpression vector, and further obtains overexpressing plants using the overexpression vector.

[0042] The steps for constructing the overexpression vector are as follows:

[0043] (1) According to the sequence shown in SEQ ID No.1, primers OEOsLBD11 / OsLBD12-F and OEOsLBD11 / OsLBD12-R were synthesized, and the primer sequences are shown in SEQ ID No.3 and SEQ ID No.4:

[0044] SEQ ID No.3, OEOsLBD11 / OsLBD12-F:

[0045] TGTACAGAGCTCGGTACCATGCGAGTCTTAGGATTAAC.

[0046] SEQ ID No.4, OEOsLBD11 / OsLBD12-R:

[0047] GTCTTTGTAGTCAAGCTTGTTTGAGCTCATTACCCCTG.

[0048] (2) Amplification of the target fragment: Extract the RNA of Nipponbare, reverse transcribe it into cDNA, use the cDNA as a template, mix the reaction system in a 50 μL centrifuge tube, and perform PCR amplification to obtain the target fragment. The reaction system is as described in Table 1.

[0049] Table 1 PCR reaction system

[0050] Reagent 50 μL reaction system 2×PhantaMax Buffer 25 μL dNTP Mix, 10 mM each 1 μL Template DNA 400 ng 10 μM Forward Primer 2 μL 10 μM Reversed Primer 2 μL PhantaMax Super-Fidelity DNA Polymerase, 1 U / μL 1 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0051] Perform gene amplification on the mixed reaction system in a PCR instrument. The conditions for the PCR reaction are set as shown in Table 2.

[0052] Table 2 PCR reaction conditions

[0053]

[0054] (3) Digestion of the expression vector with enzymes.

[0055] Perform double digestion of the required expression vector pCAMBIAI1305 with Kpn I and HindⅢ. Mix the reaction system in a 200 μL centrifuge tube and incubate in a 37 °C water bath for 3 h. The digestion system is shown in Table 3.

[0056] Table 3 Digestion system

[0057] Reagent 50 μL reaction system Plasmid vector pCAMBIAI1305 3000 ng KpnI 3 μL HindⅢ 3 μL 10×rCutSmart buffer 5 μL <![CDATA[ddH2O]]> Make up to 50 μL

[0058] (4) Obtaining the gel-extracted product.

[0059] After the reactions in (2) and (3) are completed, add 10× Loading Buffer to each tube respectively, perform electrophoresis using a 1% agarose gel by mass at a voltage of 150 V to verify the fragment size; use a gel extraction kit to recover and purify the target fragment band in the gel, and store the recovered product in a -20 °C refrigerator.

[0060] The experimental method refers to the agarose gel DNA extraction kit, which is purchased from ComWin Biotech Co., Ltd. The usage method of the agarose gel DNA extraction kit is as follows:

[0061] ① Put the cut target gel block into a 2 mL centrifuge tube, weigh it, add 1 volume of PG Buffer, and place it in a 65 °C dry bath to dissolve the gel. During this period, invert and mix continuously until it is completely dissolved, and then take it out and cool to room temperature.

[0062] ② Equilibrate the adsorption column: Add 200 μL of PS Buffer to the adsorption column and centrifuge at 12000 rpm for 1 min.

[0063] ③ Discard the waste liquid, add the gel solution to the adsorption column, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0064] ④ Discard the waste liquid, add 500 μL of PW Buffer to the adsorption column for rinsing. After centrifuging at 12,000 rpm for 1 min, rinse again.

[0065] ⑤ Discard the waste liquid, centrifuge the empty column at 12,000 rpm for 3 min to remove the residual ethanol.

[0066] ⑥ Place the adsorption column in a new 1.5 mL centrifuge tube, open the lid, and let it stand at room temperature for 5 min to volatilize the ethanol.

[0067] ⑦ Add 50 μL of preheated EB Buffer eluent at 65 °C to the adsorption column, let it stand for 1 min, and centrifuge at 12,000 rpm for 1 min to obtain the DNA solution, which is stored at -20 °C for later use.

[0068] (5) Obtain the overexpression vector by homologous recombination.

[0069] The experimental method refers to the cloning kit of Nanjing Novoprotein Scientific Co., Ltd. The steps are as follows:

[0070] ① Use DNA ligase to ligate the digested expression vector pCAMBIAI1305 and the target fragment, and mix the reaction system in a 200 μL centrifuge tube; incubate in a 37 °C water bath for 30 min to obtain the recombinant product. The ligation system is shown in Table 4.

[0071] Table 4 Ligation system

[0072] Reagent 20 μL reaction system Digested expression vector 10 μL Target gene 2 μL ExnaseⅡ 2 μL 5×Cell buffer 4 μL <![CDATA[ddH2O]]> Make up to 20 μL

[0073] ② Thaw the cloning competent cells DH5α Competent cell on ice. DH5α Competent cell is purchased from Vazyme, and the product number is #C502.

[0074] ③ Take 10 μL of the recombinant product and add it to 100 μL of the competent cells, gently flick the tube wall to mix evenly, do not shake to mix evenly, and let it stand on ice for 30 min.

[0075] ④ After heat shock in a 42 °C water bath for 45 s, immediately place it on ice to cool for 3 min.

[0076] ⑤ Add 900 μL of LB without antibiotics, shake the bacteria at 37 °C for 1 h, and the rotation speed is 250 rpm.

[0077] ⑥ Preheat the LB solid medium plate with the corresponding antibody in a 37 °C incubator.

[0078] ⑦Centrifuge at 5000 rpm and 2400 g for 5 min, and remove 900 μL of the supernatant in a laminar flow hood. After resuspending the cell pellet with the remaining medium, spread the cells evenly on a plate containing antibiotics using a sterile spreading rod.

[0079] ⑧Incubate the plate upside down in a 37 °C incubator for 16 h.

[0080] ⑨Pick a monoclonal transformant colony from the plate and transfer it to a test tube containing 5 mL of LB liquid medium with antibiotics. Tilt the test tube and incubate it in a 37 °C, 180 rpm constant temperature shaker for 12 h for expansion.

[0081] ⑩Take a part of the bacterial liquid and purify the plasmid using a plasmid miniprep kit. The plasmid miniprep kit is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number DP103. Use PCR technology and first-generation sequencing technology to verify whether the target gene is successfully and correctly ligated to the plasmid vector. If successfully constructed, mix the remaining bacterial liquid and glycerol with a volume percentage of 40% at a ratio of 1:1 and store it in a -80 °C refrigerator for long-term preservation.

[0082] The above process is as follows: Using the NIP genome as a template, amplify the gene sequence using primers SEQ ID No.3 and SEQ ID No.4. After purifying the amplification product, ligate it to the pCAMBIAI1305 vector through homologous recombination technology. Transform the ligated plasmid into Escherichia coli by heat shock method, and select positive clones for detection. After correct sequencing, send the plasmid to Wuhan Boyuan Biotechnology Co., Ltd.

[0083] The method for preparing overexpressing plants using the overexpression vector is as follows:

[0084] Using Agrobacterium tumefaciens EHA105, prepare an Agrobacterium bacterial liquid containing the above overexpression vector, and use the Nipponbare NIP strain as the background to infect the callus of rice by the Agrobacterium-mediated method; culture the infected rice to obtain two rice plants overexpressing OsLBD11 / OsLBD12, namely OsLBD11 / 12-OE1 and OsLBD11 / 12-OE2.

[0085] 2. Obtaining of rice OsLBD gene knockout mutants.

[0086] In the present invention, the target gene OsLBD is directionally knocked out by the Crispr / Cas9 technology to obtain knockout mutants of the OsLBD11 / OsLBD12 gene.

[0087] The steps for constructing the knockout vector are as follows:

[0088] First, according to the gene ID LOC_Os11g01550.1 of OsLBD11, its reference sequence in Nipponbare was downloaded. The gene ID of OsLBD12 is LOC_Os12g01550.1. Since the CDS sequences of OsLBD12 and OsLBD11 are exactly the same, materials with simultaneous mutations of the two genes were constructed.

[0089] The position of target site 1 was determined to be CGTCAGCAGCCTCGTCTACGAGG, and the position of target site 2 was CTGCAGCTGGAGTTGCAGGGCGG. Adapter primers F and adapter primer R were designed, and the primer sequences of adapter primer F and adapter primer R are shown in SEQ ID No.5 and SEQ ID No.6.

[0090] SEQ ID No.5:

[0091] cgcgctgtcgcttgtgtgGTCAGCAGCCTCGTCTACGGTTTTagagctagaaa.

[0092] SEQ ID No.6:

[0093] CTATTTCTAGCTCTAAAACCCCTGCAACTCCAGCTGCACGCCACGGAT CATCTGCA.

[0094] The construction of the rice OsLBD gene knockout lines was completed by Weimi Biotechnology Co., Ltd., and two knockout lines, oslbd11 / 12-22 and oslbd11 / 12-24, were successfully obtained.

[0095] 3. Effects of rice OsLBD knockout lines and overexpression lines on the resistance to bacterial blight.

[0096] To verify that the genes OsLBD11 / OsLBD12 are involved in the defense response of rice to bacterial blight, the changes in the resistance of OsLBD11 / OsLBD12 knockout lines and overexpression lines to bacterial blight were detected. The specific operations are as follows:

[0097] The rice bacterial blight PXO99A strain preserved in the laboratory was activated. Using NA liquid medium, it was shaken at 200 rpm and 28 °C for 12 h. The collected PXO99A bacterial cells were suspended with a 10 mM MgCl2 solution, and the OD600 was adjusted to 0.8. A sterilized scissors was dipped into the PXO99A bacterial solution and used to inoculate by cutting along the ends of the rice leaves of the OsLBD11 / OsLBD12 knockout lines and overexpression lines. The lesion lengths were measured and counted 14 days after inoculation.

[0098] The rice bacterial blight strain PXO99A was disclosed in the reference: Zeng Chen. Research on the Construction of MCP-free Mutants and Chemotactic Receptor Genes of Xanthomonas oryzae pv. oryzae [D]. Guangxi University, 2019. A The construction of MCP-free mutants and the study of chemotactic receptor genes [D]. Guangxi University, 2019.

[0099] The inoculation results are shown in Figure 1 and Figure 3 show that compared with the wild type ZH11, the lesion lengths of the knockout lines oslbd11 / 12-22 and oslbd11 / 12-24 are significantly greater than those of the wild type ZH11, indicating a significant decrease in resistance to bacterial blight. The lesion lengths of the overexpression lines OsLBD11 / 12-OE1 and OsLBD11 / 12-OE2 are significantly smaller than those of the wild type NIP, indicating that OsLBD11 / OsLBD12 is a positive regulator of rice resistance to Xanthomonas oryzae pv. oryzae. Figure 1 in A of Figure 3 in A, the position where the lesion appears is indicated by the red horizontal line upwards.

[0100] 4. Detection of the expression levels of PR genes in rice OsLBD11 / OsLBD12 knockout lines and overexpression lines after inoculation with Xanthomonas oryzae pv. oryzae.

[0101] After disinfecting the rice seeds of the wild type, knockout lines, and overexpression lines, they were cultured on 1 / 2MS solid medium for 7 days, and then rice seedlings of consistent size were selected and transferred to 1 / 2MS liquid medium for 3 days. The liquid culture medium was quickly removed, and then 1 μM Flg22 + 0.01% (v / v) surfactant and 100 μg / mL chitin + 0.01% (v / v) surfactant were added to the new liquid culture medium respectively. At the same time, an equal volume of sterile water was set as the MOCK group. After treatment for 6 h, samples were immediately taken and stored at -80 °C. Total RNA of the samples was extracted using Trizol reagent, and then 1 μg was taken for reverse transcription to synthesize cDNA using M-MLV reverse transcriptase and Olig(dT). Rice gene OsActin was used as an internal reference. Real-time fluorescence quantitative PCR was performed on rice defense-related genes, and the relative expression levels of the genes were calculated using the 2- ΔΔCT method. The results are shown in Figure 2 and Figure 4 as shown. After treatment with 1 μM Flg22 or 100 μg / mL Chitin, the expression levels of OsPR1b and OsPR10 genes in the rice overexpression lines were significantly up-regulated, while the expression levels of OsPR1b and OsPR10 genes in the rice knockout lines were significantly down-regulated, indicating that OsLBD11 / OsLBD12 can positively regulate the innate basal immune response of rice.

[0102] The real-time fluorescence quantitative PCR reaction system is shown in Table 5, the reaction conditions of the real-time fluorescence quantitative PCR reaction system are shown in Table 6, and the primer sequences of the real-time fluorescence quantitative PCR reaction are shown in Table 7.

[0103] Table 5 Real-time fluorescence quantitative PCR reaction system

[0104] Reagent 20 μL reaction system 2×FastSYBR Mixture 10 μL 10 μM Forward Primer 0.4 μL 10 μM Reversed Primer 0.4 μL Template DNA 2 μL <![CDATA[ddH2O]]> 7.2 μL

[0105] Table 6 Reaction conditions of the real-time fluorescence quantitative PCR reaction system

[0106]

[0107] Table 7 Primer sequences

[0108]

[0109]

[0110] Figure 1 and Figure 3 in, *** represents P < 0.001.

[0111] Figure 2 and Figure 4 in, different lowercase letters above the column chart indicate significant differences in gene expression levels, P < 0.05.

[0112] SEQ ID No.1:

[0113] ATGGCGGGAAGCGGGAGCGGGACACCGTGCGCGTCGTGCAAGCTGCTGCGGCGGCGGTGCACGTCGGAGTGCGTGTTCGCGCCCTACTTCCCGGCGGAGGAGGCGCAGCGGTTCGCCATGGTGCACCGGGTGTTCGGCGCCAGCAACGTCAGCAAGATGCTGCTCGATGTGCCGCCGCCGCAGAGGCCCGACGCCGTCAGCAGCCTCGTCTACGAGGCCAACGCCCGTATGAGGGACCCCGTCTACGGCTGCGTCGCCGCCATCTCCTTCCTCCAGAACCAAGTCTCCCAGCTCCAGATGCAGCTCGCCCTCGCCCACGCCGAGACCGCCGCCCTGCAACTCCAGCTGCAGCAGCAGCACCAAGATCAAGATGACCACCACCACCAGCAGTGCATCCTGGAGAATGCTGCTGCTCATCACCAGCTGATGCTGCAGGAGGCATTCCTCAAGAAAGAGTCCATGTGGACATAA。

[0114] SEQ ID No.2:

[0115] MAGSGSGTPCASCKLLRRRCTSECVFAPYFPAEEAQRFAMVHRVFGASNVSKMLLDVPPPQRPDAVSSLVYEANARMRDPVYGCVAAISFLQNQVSQLQMQLALAHAETAALQLQLQQQHQDQDDHHHQQCILENAAAHHQLMLQEAFLKKESMWT。

[0116] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0117] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of the rice OsLBD gene in enhancing the resistance of rice to bacterial blight, characterized in that, The nucleotide sequence of the OsLBD gene is shown in SEQ ID No.

1.

2. An overexpression vector, characterized in that, The overexpression vector contains the nucleotide sequence recited in Claim 1.

3. The preparation method of the overexpression vector according to claim 2, wherein, It includes the following steps: Synthesize a pair of primers, and the sequences of the primers are shown in SEQ ID No.3 and SEQ ID No.4; Extract rice RNA, reverse transcribe it into cDNA, and use the cDNA as a template to perform PCR amplification with the primers to obtain the OsLBD gene; Connect the OsLBD gene to the digested expression vector, transform it to obtain the overexpression vector.

4. The preparation method according to claim 3, wherein The expression vector includes any one of the pCAMBIA series vectors and the pBI series vectors.

5. The preparation method according to claim 4, characterized in that, The pCAMBIA series vectors include any one of pCAMBIA3301, pCAMBIA1300, and pCAMBIA1305; The pBI series vectors include any one of pBI121 and pBI101.

6. The application of the overexpression vector according to claim 2, wherein The overexpression vector is used for preparing a rice plant overexpressing the OsLBD gene, and the preparation process includes the following steps: Prepare an Agrobacterium bacterial solution containing the overexpression vector, and use the Agrobacterium bacterial solution to infect rice; Cultivate the infected rice to obtain a rice plant overexpressing the OsLBD gene.

7. The application according to claim 6, characterized in that When infecting rice, the rice variety used for infection is Nipponbare.

8. The application according to claim 6, wherein When infecting rice, use the Agrobacterium bacterial solution to infect the callus of rice.

9. The application of the overexpression vector according to claim 2, wherein The overexpression vector is used for improving the resistance of rice to bacterial blight.

Images