Application of rice transcription factor OsWRKYR1 and coding gene thereof in rice breeding for disease resistance

Abstract

The invention discloses an application of a rice transcription factor OsWRKYR1 and a coding gene thereof in rice disease-resistant breeding, and belongs to the technical field of gene engineering. The invention provides an application of a rice transcription factor OsWRKYR1 or an encoding gene thereof in any one of the following items: (1) an application in regulation and control of rice blast resistance; (2) application in improvement of rice blast resistance; and (3) application in cultivation of rice blast resistant transgenic rice. The invention proves that the over-expression of the OsWRKYR1 gene can significantly improve the resistance level of rice to rice blast, which indicates that the OsWRKYR1 can positively regulate the disease resistance of plants, and the coding gene OsWRKYR1 can be used as a target gene for improving the disease resistance of plants, has a good application prospect in the cultivation of disease-resistant rice varieties, and lays an important foundation for rice resistance breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of rice transcription factor OsWRKYR1 and its coding gene in rice disease-resistant breeding. Background Art

[0002] As an important food crop, stable and high-yield rice production is very important. However, rice blast, known as the "rice cancer", as the most devastating fungal disease, has caused serious yield losses. The current prevention and control mode relying on chemical pesticides can achieve short-term results, but long-term use is likely to trigger the evolution of pathogen drug resistance and lead to environmental risks such as soil microecological imbalance. In contrast, cultivating disease-resistant varieties, as a core prevention and control strategy with both environmental friendliness and economic benefits, can not only reduce the application of pesticides, but also is the key breakthrough for building a green prevention and control system and promoting the sustainable development of agriculture.

[0003] Based on this, focusing on the innovation of rice germplasm resources, by systematically excavating disease-resistant genetic genes with independent intellectual property rights, the technical bottleneck of the lack of existing resistance gene resources can be broken through. The obtained results can not only provide new molecular breeding targets for the prevention and control of rice blast and other fungi, but also lay a theoretical foundation and technical reserve for cultivating a new generation of high-quality and multi-resistant rice varieties. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of rice transcription factor OsWRKYR1 and its coding gene in rice disease-resistant breeding to solve the problems existing in the above-mentioned prior art. The present invention has confirmed that overexpression of the OsWRKYR1 gene can significantly improve the resistance level of rice to rice blast, indicating that OsWRKYR1 positively regulates plant disease resistance. Its coding gene OsWRKYR1 can be used as a target gene to improve plant disease resistance and has good application prospects in cultivating disease-resistant rice varieties, laying an important foundation for rice resistance breeding.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides the application of rice transcription factor OsWRKYR1 or its coding gene, and the application is any one of the following:

[0007] (1) Application in regulating the resistance of rice to rice blast;

[0008] (2) Application in improving the resistance of rice to rice blast;

[0009] (3) Application in cultivating transgenic rice resistant to rice blast;

[0010] The rice transcription factor OsWRKYR1 contains the amino acid sequence shown in SEQ ID NO.4.

[0011] Furthermore, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.3.

[0012] Furthermore, the regulation of rice blast resistance is achieved by increasing the expression level of the rice transcription factor OsWRKYR1 or its encoding gene in rice, thereby enhancing rice blast resistance.

[0013] The present invention also provides the use of a recombinant vector containing the encoding gene of the rice transcription factor OsWRKYR1, and the use is any one of the following:

[0014] (1) Use in regulating rice blast resistance;

[0015] (2) Use in enhancing rice blast resistance;

[0016] (3) Use in cultivating transgenic rice resistant to rice blast;

[0017] The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is as shown in SEQ ID NO.3.

[0018] The present invention also provides the use of a recombinant microorganism containing the encoding gene of the rice transcription factor OsWRKYR1, and the use is any one of the following:

[0019] (1) Use in regulating rice blast resistance;

[0020] (2) Use in enhancing rice blast resistance;

[0021] (3) Use in cultivating transgenic rice resistant to rice blast;

[0022] The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is as shown in SEQ ID NO.3.

[0023] The present invention also provides a method for enhancing rice blast resistance, which includes the step of stably overexpressing the encoding gene of the rice transcription factor OsWRKYR1 in rice;

[0024] The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is as shown in SEQ ID NO.3.

[0025] Furthermore, through genetic transformation technology, a recombinant vector containing the encoding gene of the rice transcription factor OsWRKYR1 is transformed into rice to achieve stable overexpression.

[0026] The present invention also provides a method for cultivating transgenic rice resistant to rice blast, which includes the step of constructing transgenic rice with stable overexpression of the encoding gene of the rice transcription factor OsWRKYR1;

[0027] The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is shown in SEQ ID NO.3.

[0028] Furthermore, through genetic transformation technology, a recombinant vector containing the encoding gene of the rice transcription factor OsWRKYR1 was transformed into rice to construct a transgenic rice with stable overexpression of the encoding gene of the rice transcription factor OsWRKYR1.

[0029] The present invention discloses the following technical effects:

[0030] The present invention identified a transcription factor induced by Magnaporthe oryzae, named OsWRKYR1. Experimental results showed that the expression levels of the OsWRKYR1 protein and its encoding gene OsWRKYR1 were closely related to the development of rice blast. By constructing overexpression and gene knockout vectors of the rice OsWRKYR1 gene, transferring the vectors into the Kitaake receptor material, obtaining OsWRKYR1 overexpression plants and knockout plants, and analyzing the disease resistance of the obtained transgenic plants, the results showed that overexpression of the OsWRKYR1 gene could significantly improve the disease resistance level of rice to rice blast, indicating that OsWRKYR1 positively regulates plant disease resistance. Its encoding gene OsWRKYR1 can be used as a target gene to improve plant disease resistance and has good application prospects in cultivating disease-resistant rice varieties, laying an important foundation for rice resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 It is a statistical chart of the expression of OsWRKYR1 in rice leaves infected with Magnaporthe oryzae and uninfected with Magnaporthe oryzae;

[0033] Figure 2Representative leaf pictures and statistical results of lesion lengths after inoculation for the control, OsWRKYR1 knockout plants, and OsWRKYR1 overexpression plants; among them, A is the representative lesion pictures of the leaves of Kitaake, oswrkyr1-KO#1, oswrkyr1-KO#2, OsWRKYR1-OE#1, and OsWRKYR1-OE#2 plants, and the scale bar is 1 cm; B is the statistical results of the lesion lengths of the inoculated plant leaves (mean ± SD, n = 24), and the asterisks above the bar graphs indicate significant differences (* indicates 0.01 < P < 0.05; ** indicates P < 0.01). Detailed implementation manners

[0034] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0038] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0039] The rice variety Kitaake is preserved by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University; it has been published in the literature "Wang Kang. Mechanism Analysis and Breeding Application Evaluation of Rice Transcription Factor OsMYB3 Regulating Blast Resistance [D]. Sichuan Agricultural University, 2019".

[0040] The rice variety Nipponbare (O. sativa L. spp. japonica, var Nipponbare, AA genome, Nip) belongs to the japonica subspecies and is recorded in the literature "Rice Variety 'Nipponbare', Agricultural Science and Technology Communication, 1973(02):31"; the rice variety Nipponbare used in this invention is provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University.

[0041] The OsWRKYR1 knockout and overexpression transgenic rice was completed by Boyuan Biotechnology Company.

[0042] The plant binary expression vector pYLCRISPR / Cas9Pubi-H is provided by Associate Professor Tang Yongyan of the State Key Laboratory of Sichuan Agricultural University. Its construction method is published in the literature "A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants [J]. Molecular Plant, 2015, 8(8):1274-1284".

[0043] The plant binary expression vector pRHVcMyc is provided by Professor Chen Xuewei of the State Key Laboratory of Sichuan Agricultural University, and its construction method is published in the literature "A Versatile Vector Toolkit for Functional Analysis of Rice Genes [J]. Rice,

[0044] 2018, 11(1):27".

[0045] Total RNA extraction kit: TRIzol purchased from Invitrogen, USA, with the product number 15596026.

[0046] Reverse transcription kit: HiScript III RT SuperMix for qPCR(+gDNAwiper) purchased from Vazyme, China, with the product number R323-01.

[0047] Homologous recombination kit: ClonExpress II One Step Cloning Kit purchased from Vazyme, China, with the product number C112-01.

[0048] In the study of rice immune response, a transcription factor induced by Magnaporthe oryzae was identified and named OsWRKYR1. OsWRKYR1 plays an important role in plants' response to abiotic stresses. Therefore, previous studies on OsWRKYR1 focused on its regulation of phosphate response and cold stress. The research of this invention shows that OsWRKYR1 knockout plants are more susceptible to M. oryzae, while OsWRKYR1 overexpressing plants are more resistant to M. oryzae, indicating that OsWRKYR1 positively regulates rice resistance to M. oryzae and can be used as a target gene for molecular breeding to improve plant disease resistance.

[0049] Example 1 Rice transcription factor OsWRKYR1 and its cloning

[0050] A transcription factor OsWRKYR1 was screened in rice. According to the reference gene sequence of the rice Nip genome, the following amplification primers were designed:

[0051] OsWRKYR1-F: ATGGAGAGCATGGAGGGCAA (SEQ ID NO.1);

[0052] OsWRKYR1-R: TCATGCGAAGAAGCTGGTGA (SEQ ID NO.2).

[0053] Using Kitaake total cDNA as a template, a DNA band of about 1.1 kb was amplified by the above primers. After sequencing, it was confirmed to be the OsWRKYR1 gene belonging to the transcription factor.

[0054] The nucleotide sequence of OsWRKYR1 is shown in SEQ ID NO.3:

[0055]

[0056] The amino acid sequence of the OsWRKYR1 protein is shown in SEQ ID NO.4:

[0057] MESMEGNGGGRLVVTELSHIKELVRQLEGHLGGSGSPDLCKHLASQIFSVTERSIGMIRSGHFDGHRKRSAAAVAAGDLDSATPSPLSDVSDLPFKATKKRKTSTEKKRHQIRVSSTGGVENPPVDDGHSWRKYGQKEILGAKHPRGYYRCTHRHSQGCMATKQVQRTDEDATVFDVIYHGEHTCVHKAVAAGAGKPETETDTNAAAESRLHDLSSGLTVKIEGLTAPPQQQQGGGGWNAMPPFCLSSPVSGLAPPDQHNPFSAPSTPENRLAAAASSAASPATSDSMAAAPFHQAAAGGGDEAWRDAELQEVVSALVAATTTTATAQPAPATAMVDADLSALDAFEFDPGFTIDITSFFA*。

[0058] The following real-time quantitative PCR primers were designed according to the nucleotide sequence of OsWRKYR1:

[0059] F: AGGCATCAGATTAGGGTGAGC (SEQ ID NO.5);

[0060] R: GGTGGGTGCAACGGTAGT (SEQ ID NO.6).

[0061] To verify the potential function of OsWRKYR1 in rice, rice material cultivation and rice blast fungus infection experiments were carried out as follows:

[0062] (1) The experimental rice material was Kitaake, a rice blast-susceptible variety. The Kitaake material was planted in the field until the three-leaf and one-heart stage.

[0063] (2) The tested rice blast fungus was the spores of Magnaporthe oryzae Zhong10-8-14 (published in the literature "A single transcription factor promotes both yield and immunity in rice[J].Science,2018,361(6406):1026-1028."). The spray inoculation method was used to inoculate the concentration of 5×10 5The spores of Magnaporthe oryzae were inoculated onto Kitaake leaves. The rice leaves at 0 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, and 72 h after inoculation were quickly frozen in liquid nitrogen and stored in a -80 °C refrigerator. Total RNA of rice was extracted using Trizol reagent and reverse transcribed into cDNA. Using the above real-time quantitative PCR primers, the expression of the OsWRKYR1 gene in rice leaves infected with Magnaporthe oryzae (Zhong10-8-14) and rice leaves not infected with the pathogen (Mock, replacing the pathogen with an equal amount of 0.1% Tween-20) was detected.

[0064] The results are as Figure 1 shown. After infection with Magnaporthe oryzae, the expression of the OsWRKYR1 gene in Kitaake was significantly upregulated, indicating that this gene plays an important role in the development of rice blast.

[0065] Example 2 Construction of the OsWRKYR1 plant knockout vector

[0066] Using the genome of rice Nip as a template, the knockout target sequence was designed as: ACTTGCCTTTCAAGGCCACC (SEQ ID NO.7).

[0067] According to the target, the following primers were designed and synthesized:

[0068] F: TGTGACTTGCCTTTCAAGGCCACCG (SEQ ID NO.8);

[0069] R: AAAACGGTGGCCTTGAAAGGCAAGT (SEQ ID NO.9).

[0070] After denaturation at 95 °C, DNA double strands were formed by annealing. The pYLCRISPR / Cas9Pubi-H vector was digested with BsaI, and the linearized vector was recovered. The above DNA double strands and the linearized vector were mixed according to the following system (1 μL of DNA double strand fragment, 1 μL of linearized vector, 1 μL of T4 ligase, 1 μL of T4 ligase buffer, 6 μL of ddH2O, total volume 10 μL), and incubated at room temperature for 30 minutes for T4 DNA ligase ligation reaction to obtain the pYLCRISPR / Cas9Pubi-H-sgRNA OsWRKYR1 knockout vector. It was transformed into DH5α competent cells. After screening with kanamycin, single colonies were picked for colony PCR identification. After the positive colonies were extracted with plasmids and sequenced correctly, it was confirmed that the pYLCRISPR / Cas9Pubi-H-sgRNA OsWRKYR1 was correct and could be used for genetic transformation.

[0071] The genetic transformation of the knockout vector pYLCRISPR / Cas9Pubi-H-sgRNA was completed by Boyuan Biotechnology Company, and the OsWRKYR1 knockout transgenic rice oswrkyr1-KO#1 and oswrkyr1-KO#2 were obtained. OsWRKYR1

[0072] Example 3 Construction of OsWRKYR1 Plant Overexpression Vector

[0073] Using the cDNA reverse transcribed from the total RNA of rice Nip as a template, the following primers for cloning OsWRKYR1 were designed:

[0074] F: ATCCAGATCCAGTGGGATCC ATGGAGAGCATGGAGGGCAA (SEQ ID NO.10);

[0075] R: GTAAGCTTGGTACCGAGCTCTGCGAAGAAGCTGGTGATAT (SEQ ID NO.11).

[0076] The PCR reaction was carried out on a PE9600 type PCR instrument, and its program was: pre-denaturation at 94 °C for 2 min; then denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 72 °C for 2 min, for a total of 30 - 32 cycles; then extension at 72 °C for 10 min; and preservation at 4 °C. The full-length cDNA of OsWRKYR1 was obtained by PCR amplification. The pRHVcMyc vector was digested with BamHI and SacI double enzymes, and the linearized vector was recovered. The above PCR recovered product, the full-length cDNA of OsWRKYR1 and the linearized vector were mixed according to the following system (2 μL of DNA fragment, 2 μL of linearized vector, 2 μL of 5×CE II Buffer, 1 μL of Exnase II and 3 μL of ddH2O, with a total volume of 10 μL), and homologous recombination was carried out at 37 °C for 30 min to obtain the pRHVc-OsWRKYR1-Myc recombinant vector.

[0077] The recombinant vector was transformed into DH5α competent cells. After screening with kanamycin, single colonies were picked for colony PCR identification. After the positive colonies were extracted with plasmids and the sequencing was correct, it was confirmed that the pRHVc-OsWRKYR1-Myc overexpression vector was correct and could be used for genetic transformation.

[0078] The genetic transformation of the pRHVc-OsWRKYR1-Myc overexpression vector was completed by Boyuan Biotechnology Company, and the OsWRKYR1 overexpression transgenic rice OsWRKYR1-OE#1 and OsWRKYR1-OE#2 were obtained.

[0079] Example 4 OsWRKYR1 Is Involved in Regulating Plant Disease Resistance ​

[0080] In plants, OsWRKYR1 mainly plays a role in the response of plants to abiotic stresses. The present invention discovers that OsWRKYR1 plays an important role in regulating the disease resistance response of plants. Therefore, the following experiments were conducted to test the effect of the gene OsWRKYR1 on the disease resistance of rice:

[0081] Wounding inoculation treatment at the seedling stage: The test materials included Kitaake, the OsWRKYR1 knockout lines oswrkyr1-KO#1 and oswrkyr1-KO#2 with a stable inheritance background of Kitaake, and the OsWRKYR1 overexpression lines OsWRKYR1-OE#1 and OsWRKYR1-OE#2 with a stable inheritance background of Kitaake. Select seeds with plump grain shapes and place them in a conical flask filled with tap water. Germinate them in a 37°C dark incubator and change the tap water every day. After 2 days, select the seeds with germinated tips and place them in a 96-well seedling plate. Place the 96-well seedling plate on a floating raft and grow them in Hoagland nutrient solution. After 21 days, select the second-to-last rice leaves with consistent growth and size. After wounding, inoculate 5 μL of the spores of Magnaporthe oryzae Zhong10-8-14 with a concentration of 3×10 5 / ml. Observe and count the lesion lengths after 5 days. The results are as Figure 2 shown. The statistical results show that after wounding inoculation, compared with Kitaake, the lesion lengths of the plants of oswrkyr1-KO#1 and oswrkyr1-KO#2 with a Kitaake background are extremely significantly longer and the disease resistance is weakened. Compared with Kitaake, the lesion lengths of the plants of OsWRKYR1-OE#1 and OsWRKYR1-OE#2 with a Kitaake background are extremely significantly reduced and the disease resistance of the plants is enhanced. It shows that OsWRKYR1 positively regulates the disease resistance of plants, and its coding gene OsWRKYR1 can be used as a target gene for molecular breeding to improve the disease resistance of plants.

[0082] The embodiments described above are only used to describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of the rice transcription factor OsWRKYR1 or its encoding gene, characterized in that, The application is any one of the following: (1) Application in regulating rice blast resistance; (2) Application in enhancing rice blast resistance; (3) Application in cultivating transgenic rice resistant to rice blast; The rice transcription factor OsWRKYR1 contains the amino acid sequence shown in SEQ ID NO.

4.

2. The application according to claim 1, characterized in that The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

3.

3. The application according to claim 1, characterized in that, The regulation of rice blast resistance is achieved by increasing the expression level of the rice transcription factor OsWRKYR1 or its encoding gene in rice, thereby enhancing rice blast resistance.

4. Use of a recombinant vector containing the gene encoding the rice transcription factor OsWRKYR1, characterized in that, The application is any one of the following: (1) Application in regulating rice blast resistance; (2) Application in enhancing rice blast resistance; (3) Application in cultivating transgenic rice resistant to rice blast; The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is shown in SEQ ID NO.

3.

5. Use of a recombinant microorganism containing the gene encoding the rice transcription factor OsWRKYR1, characterized in that, The application is any one of the following: (1) Application in regulating rice blast resistance; (2) Application in enhancing rice blast resistance; (3) Application in cultivating transgenic rice resistant to rice blast; The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is shown in SEQ ID NO.

3.

6. A method for improving the blast resistance of rice, characterized in that, It includes the step of stably overexpressing the encoding gene of the rice transcription factor OsWRKYR1 in rice; The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is shown in SEQ ID NO.

3.

7. The method according to claim 6, characterized in that Through genetic transformation technology, a recombinant vector containing the encoding gene of the rice transcription factor OsWRKYR1 is transformed into rice to achieve its stable overexpression.

8. A method for cultivating transgenic rice resistant to rice blast, characterized in that, It includes the step of constructing a transgenic rice with stable overexpression of the encoding gene of the rice transcription factor OsWRKYR1; The nucleotide sequence of the encoding gene of the rice transcription factor OsWRKYR1 is shown in SEQ ID NO.

3.

9. The method according to claim 8, characterized in that Through genetic transformation technology, a recombinant vector containing the encoding gene of the rice transcription factor OsWRKYR1 is transformed into rice to construct a transgenic rice with stable overexpression of the encoding gene of the rice transcription factor OsWRKYR1.

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