Method for improving disease resistance of rice through double knockout transcription factor genes OsWRKY62 and OsWRKY76

Abstract

The invention belongs to the field of crop genetic breeding, and discloses a method for improving disease resistance of rice through double knockout transcription factor genes OsWRKY62 and OsWRKY76. The nucleotide sequences of the OsWRKY62 gene and the OsWRKY76 gene are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2, and the amino acid sequences of the encoding proteins of the OsWRKY62 gene and the OsWRKY76 gene are shown as SEQ ID NO. 3 and SEQ ID NO. 4. It is found that rice plants become short due to respective knockout or double knockout of the two genes, the double knockout phenotype is obvious, and the mutated genes can improve the resistance of rice to bacterial blight and rice blast. The synergistic effect of the two genes provides a new genetic resource for rice bacterial leaf blight and rice blast resistance breeding research.

Description

Technical Field

[0001] The present invention belongs to the field of crop genetic breeding, and specifically relates to a method for knocking out transcription factor genes. OsWRKY62 and OsWRKY76 Methods to improve disease resistance in rice. Background Art

[0002] As one of the most important food crops in the world, rice (Oryza sativa) has long faced severe challenges from diseases. Therefore, improving rice's disease resistance is considered a key strategy to ensure its stable production. As an important type of transcription factor in plants, the WRKY gene family has attracted much attention due to its core regulatory role in plant disease resistance signal transduction pathways and has become a focus of molecular plant pathology research. This family has many members, and a large number of WRKY members have been successfully cloned in different plants. In particular, more than 100 WRKY genes have been identified in rice. Despite this, to date, only a few WRKY genes have been clearly verified for their functions. WRKY transcription factors construct complex regulatory networks, enabling plants to quickly adjust their internal physiological states when attacked by pathogens, achieving a delicate balance between effectively resisting pathogen invasion and avoiding excessive defense responses that have a negative impact on their own growth and development. In-depth analysis of the functions of WRKY transcription factors and the construction of their regulatory networks can not only provide valuable information for studying the co-evolution of pathogens and host plant defense systems, but also help discover new functional gene combinations that act in the same biological process or signaling pathway. This research result is of great significance for revealing the mechanism of gene cooperation that has not been touched by traditional genetic methods. Summary of the invention

[0003] The present invention aims to provide two new rice disease resistance genes, namely mutant rice WRKY genes oswrky62 and oswrky76 (members of the WRKY transcription factor family), the synergistic effect of these two genes can be applied to the study of rice resistance mechanism and resistance breeding. Single knockout and double knockout of the two genes involved in the present invention will cause the plant to become shorter and improve resistance, among which the double knockout mutant phenotype is more significant and has stronger resistance to bacterial blight and rice blast.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme: Rice WRKY genes OsWRKY62 and OsWRKY76 Double knockout mutant oswrky62 / oswrky76 , the rice WRKY gene OsWRKY62 The nucleotide sequence of the rice WRKY gene is shown in SEQ ID NO.1; OsWRKY76The nucleotide sequence is shown in SEQ ID NO.2.

[0005] The above rice WRKY genes OsWRKY62 The amino acid sequence of the encoded protein is shown in SEQ ID NO.3; the rice WRKY gene OsWRKY76 The amino acid sequence of the encoded protein is shown in SEQ ID NO.4.

[0006] The present invention also provides the rice WRKY gene OsWRKY62 and OsWRKY76 Double knockout mutant oswrky62 / oswrky76 Application in improving disease resistance of rice.

[0007] The disease resistance is rice bacterial blight and / or rice blast.

[0008] A method for improving rice disease resistance, comprising the steps of obtaining a mutant by double knocking out the above rice WRKY gene oswrky62 / oswrky76 .

[0009] The beneficial effects of the present invention are: rice WRKY gene OsWRKY62 and OsWRKY76 The synergistic effect after double knockout provides new genetic resources for breeding research on rice resistance to bacterial blight and blast.

[0010] This project used MH86 as the background material and obtained two homozygous WRKY62 Single knockout strains oswrky62-1,oswrky62-2 , 2 homozygous WRKY76 Single knockout strains oswrky76-1,oswrky76-2 and 2 homozygous WRKY62、WRKY76 Double knockout strains oswrky62-1 / oswrky76-1 , oswrky62-2 / oswrky76-2 Phenotypic analysis showed that the mutant plants were shorter than the wild type, and the phenotype of the double knockout mutant was more obvious than that of the single knockout mutant. WRKY62 and WRKY76 Double knockout mutants will improve rice disease resistance. Exploring the functions of these two genes and the signal pathways they may be involved in will enrich the theoretical basis for plants to produce PCD and improve resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :MH86 OsWRKY62 Schematic diagram of mutation types; (A) mutation site; (B) mutation type.

[0013] Figure 2:MH86 OsWRKY76 Schematic diagram of mutation types; (A) mutation site; (B) mutation type.

[0014] Figure 3 :MH86 OsWRKY62 / OsWRKY76 Schematic diagram of mutation types.

[0015] Figure 4 : Phenotypes of wild-type MH86 and mutant plants.

[0016] Figure 5 : Double knockout oswrky62-1 / oswrky76-1 Improve rice resistance to bacterial blight.

[0017] Figure 6 : Double knockout oswrky62-1 / oswrky76-1 Improve rice resistance to rice blast. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0019] The technical means described in the embodiments are conventional means familiar to those skilled in the art and will not be described in detail.

[0020] MH86 in the examples was obtained from our research group; the bacterial blight pathogen was obtained from the Rice Research Institute of Fujian Academy of Agricultural Sciences; and the rice blast pathogen was obtained from the Institute of Plant Protection of Fujian Academy of Agricultural Sciences.

[0021] The composition of YEB medium: 5g sucrose, 10g tryptone, 5g yeast powder, 0.5g MgSO4·7 H2O, 2% agar, pH7.2-7.4, and sterile water to make up to 1L.

[0022] The composition of starch yeast culture medium: 10 g soluble starch, 2 g yeast extract, 18 g agar, pH value is 6.0-6.5, and the volume is made up to 1L with sterile water.

[0023] The composition of rice bran culture medium: 20g rice bran, 2.5g yeast extract, 18g agar, pH value is 6.0-6.5, and the volume is made up to 1L with sterile water.

[0024] Example 1 WRKY62、WRKY76 Preparation of single and double knockout mutant materials Respectively WRKY62、WRKY76The gene accession number was logged into the CRISPR-GE (http: / / skl.scau.edu.cn / home / ) website for prediction of CRISPR / Cas9 knockout targets. At the same time, appropriate target sequences were selected as gRNA targets in the front section of the coding region and the conserved region of protein kinases. The target sequences were then logged into the CRISPRRGEN Tools (http: / / www.rgenome.net / cas-offinder / ) website for off-target analysis, and the targets were introduced by PCR amplification. Finally, the CRISPR / Cas9 knockout vector of the pHUE411 gene was constructed using the Golden Gate cloning method. After correct sequencing, the recombinant plasmid was transformed into Agrobacterium (EHA105) competent cells using the freeze-thaw method. With MH86 as the background material, transgenic materials were obtained through Agrobacterium-mediated rice genetic transformation. After DNA was extracted from the obtained T0 generation transgenic plants, CRISPR / Cas9 vector-specific primers were used to detect whether they were positive plants. The primer sequences are as follows: OsU3-FD3: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' TaU3-RD: 5'-CTCACAAATTATCAGCACGCTAGTC-3' The positive plants were planted in the experimental field, and the T1 seedlings were harvested and leaves were taken for DNA extraction. WRKY62, WRKY76 Primers were designed near the two target sites and four target sites of the single knockout mutant and double knockout mutant to detect the knockout status. After testing, two homozygous WRKY62 Single knockout strains oswrky62-1,oswrky62-2 , 2 homozygous WRKY76 Single knockout strains oswrky76-1,oswrky76-2 and 2 homozygous WRKY62、WRKY76 Double knockout strains oswrky62-1 / oswrky76-1 , oswrky62-2 / oswrky76-2 The plants were grown in the field, and their phenotypes were observed and photographed. Figure 4 As shown, compared with the wild type, the mutant plants became shorter, and the phenotype of the double knockout mutant was more obvious than that of the single knockout mutant.

[0025] Example 2 OsWRKY62、OsWRKY76 Gene negatively regulates rice resistance to bacterial blight The bacterial blight pathogen was cultured in YEB solid medium at 28°C for 2-3 days, and the bacteria were washed with sterile water to adjust the OD of the bacterial blight suspension. 600 to 0.5, and then dipped the suspension of Xanthomonas solani with sterile scissors and applied it to wild-type rice (WT), oswrky62-1、oswrky76-1 and oswrky62-1 / oswrky76-1The fully expanded leaves of the material were cut at the same position to inoculate the bacterial blight pathogen. The results were investigated and counted after 15 days. Figure 5 As shown, WRKY62 and WRKY76 Single gene knockout ( oswrky62-1、oswrky76-1 ) and double knockout homozygous materials ( oswrky62-1 / oswrky76-1 ) increased resistance to bacterial blight, and double knockout oswrky62-1 / oswrky76-1 Stronger resistance to bacterial blight.

[0026] Example 3 OsWRKY62、OsWRKY76 Gene negatively regulates rice resistance to rice blast fungus Inoculate the filter paper containing the rice blast fungus strain "SH19029-1" into the center of the starch yeast culture medium plate, place it in a 26°C biochemical incubator and invert it for culture for 5-6 days; pick up freshly grown mycelium blocks and inoculate them onto the rice bran culture medium plate, culture it at a constant temperature of 26°C for 7-8 days, and when the mycelium fills the culture dish, use a sterile glass slide to scrape off the aerial mycelium, and then place it in a 25°C constant temperature light incubator for light spore production for 2-3 days, then collect conidia and prepare a spore suspension for inoculation.

[0027] When the rice seedlings grow to the three-leaf and one-heart stage, use an air compressor connected to a throat sprayer to spray and inoculate. Use a Tween 20 solution with a final concentration of 0.02% to prepare a suspension of conidia of rice blast fungus, and the final concentration of spores is 1.5×10 5 / mL. After inoculation, place in a greenhouse at a constant temperature and humidity of 22-23 ℃ for 24 hours in the dark, then remove and place in a constant temperature greenhouse with a humidity higher than 80% for moisturizing culture. 5-7 days after inoculation, investigate the disease of rice. The visual method is used to refer to the international rice leaf scar grading standard to investigate and record the type of lesion reaction on each rice leaf. If different lesion reaction types appear on the same leaf, the highest reaction type shall be used as the standard. The results are as follows Figure 6 As shown, single knockout ( oswrky62-1 , oswrky76-1 ) and double knockout homozygous materials ( oswrky62-1 / oswrky76-1 ) can improve resistance to rice blast fungus, and double knockout oswrky62-1 / oswrky76-1 Stronger resistance to rice blast fungus.

[0028] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. Rice WRKY genes OsWRKY62 and OsWRKY76 Double knockout mutant oswrky62 / oswrky76 , characterized in that: The rice WRKY gene OsWRKY62 The nucleotide sequence of the rice WRKY gene is shown in SEQ ID NO.1; OsWRKY76 The nucleotide sequence is shown in SEQ ID NO.

2.

2. The rice WRKY gene according to claim 1 OsWRKY62 and OsWRKY76 Double knockout mutant oswrky62 / oswrky76 , characterized in that: The rice WRKY gene OsWRKY62 The amino acid sequence of the encoded protein is shown in SEQ ID NO.3; the rice WRKY gene OsWRKY76 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

4.

3. The rice WRKY gene according to claim 1 OsWRKY62 and OsWRKY76 Double knockout mutant oswrky62 / oswrky76 Application in improving rice disease resistance.

4. The use according to claim 3, characterized in that: The disease resistance is rice bacterial blight and / or rice blast.

5. A method for improving disease resistance of rice, characterized in that: By double knocking out the rice WRKY gene as claimed in claim 1 OsWRKY62 and OsWRKY76 .

Images