Application of Rice E3 Ubiquitin Ligase BISG1 and Its Encoding Gene in Rice Disease Resistance Breeding

Abstract

The invention discloses the application of rice E3 ubiquitin ligase BISG1 and its encoding gene in rice disease resistance breeding, belongs to the field of gene engineering technology, and discloses the application of rice E3 ubiquitin ligase BISG1 in regulating rice blast resistance, improving rice blast resistance or cultivating transgenic rice resistant to rice blast. The invention constructs overexpression and gene knockout vectors of rice BISG1 gene, transfers the vectors into Taipei 309 receptor material, obtains BISG1 overexpression plants and knockout plants, and analyzes the disease resistance of the obtained transgenic plants. The results show that knocking out BISG1 gene significantly improves the resistance level of rice to rice blast, indicating that BISG1 negatively regulates the disease resistance of plants, and its encoding gene BISG1 can be used as a target gene to improve plant disease resistance, has good application prospects in cultivating disease-resistant rice varieties, and lays an important foundation for rice resistance breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to application of rice E3 ubiquitin ligase BISG1 and a coding gene thereof in rice disease resistance breeding. Background Art

[0002] Fungal diseases such as rice blast cause large-scale crop yield reduction, so breeding disease-resistant crops is one of the main goals of the planting industry. The prior art has reported a number of genes related to plant disease resistance, including effector molecule genes and regulatory genes, which are derived from crops such as rice, wheat, corn, soybeans, and model plants such as Arabidopsis. Some of them have been used as target genes in crop disease resistance genetic engineering, and disease-resistant rice, wheat, corn, soybeans, etc. have been successfully cultivated. However, due to differences in genetic background, some genes have application barriers, so new related genes are still needed to meet the needs of breeding.

[0003] As one of the most important food crops, improving the disease resistance of rice has important theoretical and practical significance. To this end, the present invention mines new disease-resistant genetic resources for improving the resistance level of rice blast, providing an effective and economical strategy for disease-resistant rice breeding. Summary of the invention

[0004] The purpose of the present invention is to provide the application of rice E3 ubiquitin ligase BISG1 and its encoding gene in rice disease resistance breeding to solve the problems existing in the above-mentioned prior art. The present invention confirms that knocking out the BISG1 gene significantly improves the resistance level of rice to rice blast, indicating that BISG1 negatively regulates the disease resistance of plants. Its encoding gene BISG1 can be used as a target gene to improve plant disease resistance. It has good application prospects in cultivating disease-resistant rice varieties and lays an important foundation for rice resistance breeding.

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

[0006] The present invention provides an application of rice E3 ubiquitin ligase BISG1, wherein the application comprises any one of the following:

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

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

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

[0010] The rice E3 ubiquitin ligase BISG1 comprises the amino acid sequence shown in SEQ ID No.2.

[0011] The present invention also provides the use of the gene encoding the rice E3 ubiquitin ligase BISG1 in any of the following items:

[0012] (1) Application in regulating rice blast resistance;

[0013] (2) Application in improving rice resistance to rice blast;

[0014] (3) Application in breeding transgenic rice resistant to rice blast;

[0015] The coding gene comprises the nucleotide sequence shown as SEQ ID No.1.

[0016] The present invention also provides the use of a recombinant vector comprising the above encoding gene in any of the following:

[0017] (1) Application in regulating rice blast resistance;

[0018] (2) Application in improving rice resistance to rice blast;

[0019] (3) Application in breeding transgenic rice resistant to rice blast.

[0020] The present invention also provides the use of a recombinant bacterium comprising the above-mentioned recombinant vector in any of the following items:

[0021] (1) Application in regulating rice blast resistance;

[0022] (2) Application in improving rice resistance to rice blast;

[0023] (3) Application in breeding transgenic rice resistant to rice blast.

[0024] Furthermore, the resistance of rice to rice blast is improved by down-regulating the expression level of the rice E3 ubiquitin ligase BISG1 or its encoding gene.

[0025] Furthermore, a knockout vector of the encoding gene is constructed using the CRISPR / Cas9 method, and the knockout vector is transferred into a rice recipient material to improve the resistance of rice to rice blast.

[0026] The present invention also provides a method for improving rice blast resistance, comprising the step of down-regulating the expression level of a rice E3 ubiquitin ligase BISG1 encoding gene; the nucleotide sequence of the rice E3 ubiquitin ligase BISG1 encoding gene is shown in SEQ ID No.1.

[0027] Furthermore, a knockout vector of the rice E3 ubiquitin ligase BISG1 encoding gene was constructed using the CRISPR / Cas9 method, and the knockout vector was transferred into a rice recipient material to improve the rice resistance to rice blast.

[0028] The present invention discloses the following technical effects:

[0029] The present invention identifies an E3 ubiquitin ligase induced by rice blast fungus, and names it BISG1. Experimental results show that the expression of BISG1 protein and its encoding gene BISG1 are closely related to the development of rice blast disease. The present invention constructs an overexpression and gene knockout vector of rice BISG1 gene, transfers the vector into Taipei 309 (TP309) receptor material, obtains BISG1 overexpression plants and knockout plants, and analyzes the disease resistance of the obtained transgenic plants. The results show that knocking out the BISG1 gene significantly improves the resistance level of rice to rice blast disease, indicating that BISG1 negatively regulates the disease resistance of plants, and its encoding gene BISG1 can be used as a target gene to improve plant disease resistance, has good application prospects in cultivating disease-resistant rice varieties, and lays an important foundation for rice resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 The expression statistics of BISG1 in rice leaves before and after rice blast infection; The figure shows the expression changes of BISG1 in leaves of three-week-old Taipei 309 (TP309) plants after being inoculated with rice blast spores (physiological subspecies zhong10-8-14) and Mock (0.1% Tween-20) for 0h, 0.5h, 1h, 3h, 6h, and 12h; the asterisk above the bar graph indicates a significant difference (P<0.01, n=3);

[0032] Figure 2 Figure 1 is the phenotype and statistical results of the control and BISG1 knockout plants after inoculation with puncture wounds; A is a representative picture of lesions on leaves of three-week-old Taipei 309 (TP309), bisg1-ko#1 (BISG1-KO1) and bisg1-ko#2 (BISG1-KO2) plants 5 days after being inoculated with rice blast fungus (physiological subspecies zhong10-8-14), with a scale of 1 cm; B is the statistical result of the lesion length on leaves of plants inoculated with puncture wounds (mean ± SD, n = 20), and the asterisk above the bar graph indicates a significant difference (P < 0.01);

[0033] Figure 3Figure 2 shows the phenotypes and statistical results of the control and BISG1 overexpressing plants after being inoculated with blast fungus. Figure 3 shows the representative lesion pictures of leaves of three-week-old Taipei 309 (TP309), BISG1-OE#1 (BISG1-OE1) and BISG1-OE#2 (BISG1-OE2) plants 5 days after being inoculated with rice blast fungus (physiological subspecies zhong10-8-14). The scale bar is 1 cm. Figure 4 shows the statistical results of the lesion length of leaves of plants inoculated with blast fungus after being inoculated with blast fungus (mean ± SD, n = 20). The asterisks above the bar graphs indicate significant differences (P < 0.01). DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The rice variety Taipei 309 is maintained by Professor Xuewei Chen of the State Key Laboratory of Sichuan Agricultural University.

[0040] The rice variety Nipponbare (O. sativa L. spp. japonica, var Nipponbare, AA genome, Nip) belongs to the japonica subspecies, as recorded in the following literature: Rice variety "Nipponbare", Agricultural Science and Technology Communications, 1973(02):31; provided by Professor Chen Xuewei of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0041] BISG1 knockout and overexpression transgenic rice were produced by Boyuan Biotechnology Company.

[0042] The plant binary expression vector pTCRISPR was provided by Associate Professor Tang Yongyan from the State Key Laboratory of Sichuan Agricultural University.

[0043] The plant binary expression vector pRHVc-Ubi-HA-nosT was provided by Professor Xuewei Chen from the State Key Laboratory of Sichuan Agricultural University.

[0044] Total RNA extraction kit: TRIzol purchased from Invitrogen, USA, catalog number 15596026.

[0045] Reverse transcription kit: HiScript III RT SuperMix for qPCR (+gDNA wiper) purchased from Vazyme, China, with the catalog number R323-01.

[0046] Homologous recombination kit: ClonExpress II One Step Cloning Kit purchased from Vazyme, China, with the catalog number C112-01.

[0047] In the study of rice immune response, the present invention identified an E3 ubiquitin ligase induced by rice blast fungus and named it BISG1. BISG1 plays an important role in plant response to abiotic stress, so previous studies on BISG1 focused on its regulation of low phosphorus response and drought stress. The research of the present invention shows that BISG1 knockout plants are more resistant to rice blast, while BISG1 overexpression plants are more susceptible to rice blast, indicating that BISG1 negatively regulates rice resistance to rice blast and can be used as a target gene for molecular breeding to improve plant disease resistance. The specific research is as follows:

[0048] Example 1 Rice E3 ubiquitin ligase BISG1 and its cloning

[0049] An E3 ubiquitin ligase BISG1 was screened in rice, and primers were designed according to the reference gene sequence of the rice Nip genome:

[0050] BISG1F: ATGGCGCTGCTGGCGCGGAGGG;

[0051] BISG1R: TCATGACAGCCTATTGAGCCCT.

[0052] Using TP309 total cDNA as a template, the above primers were used to amplify a DNA band of about 1.3 kb, which was confirmed to belong to the BISG1 gene of the E3 ubiquitinase family after sequencing.

[0053] The nucleotide sequence of the gene BISG1 amplified from the total cDNA of TP309 is shown in SEQ ID No. 1:

[0054]

[0055] The amino acid sequence of BISG1 protein is shown in SEQ ID No.2:

[0056] MALLARRARKAVMAKAPAPLLQKRGGGAAAELAIPAHFRCPISLMRDPVTAPTGITYDREGIEAWLDTGRAVCPVTHAPLRHEDLVPNHAIRRVIQDWCVANRSRGVERIPTPKIPVTPVQASELLFDVAESAARRGAAGRAAGAVARVRALARDSERNRRCFVSVGTGRVLAAAFESLAAAGEAGVLEDVLAALVCMMPLDEEAARVLASSSSMGSLVAIAK HGSLAGRLNAVLAIKEAVSRDGAFVDLADDKVDKVVDALVVIIKAPICPQATKAAMVATYHLASSDERVAARVASTGLVPTLIEALVDADKSVSEKALAVLDAMLASEEGRA SARGHALAMPALVKKMFRVSDVATELAVSAMWRLGCKASSGDEEAAATGCLVEALRVGAFQKLLLLLQVGCRDATKEKATELLKMLNKHKGLGECVDAVDFRGLNRLS*(SEQ ID No.2).

[0057] The following real-time quantitative PCR primers were designed based on the nucleotide sequence of BISG1:

[0058] F:GAAGCTTCTCCTCCTCCTGC;

[0059] R:AGCCCTCTTGAAATCCACAGC.

[0060] Material culture and rice blast pathogen infection

[0061] (1) The experimental rice material was Taipei 309 (TP309), a rice blast susceptible variety. TP309 was planted in the field until the three-leaf stage.

[0062] (2) The pathogen of rice blast was Zhong10-8-14 rice blast spores. 5 μL of 5×10 5 ml -1Rice blast fungus spores were inoculated into TP309 leaves at the tillering stage. Rice leaves were taken at 0h, 0.5h, 1h, 3h, 6h and 12h after inoculation, frozen in liquid nitrogen and stored in a -80℃ refrigerator. Total RNA of rice was extracted using Trizol reagent and reverse transcribed to obtain cDNA for real-time quantitative PCR detection. The results are shown in Figure 1 As shown, the expression of BISG1 gene in TP309 was significantly upregulated after infection by rice blast fungus, indicating that this gene plays an important role in the development of rice blast disease.

[0063] Example 2 Construction of BISG1 plant knockout vector

[0064] BISG1 knockout vector pTCRISPR-sgRNA BISG1

[0065] Using the genome of rice Nip as a template, the knockout target sequence was designed as: GAGGGCGCGGAAGGCGGTGA.

[0066] The following primers were synthesized:

[0067] F:TGTG GAGGGCGCGGAAGGCGGTGA G;

[0068] R:AAAAC TCACCGCCTTCCGCGCCCTC.

[0069] After denaturation at 95°C, anneal to form a double-stranded DNA. The pTCRISPR vector was digested with BsaI to recover the linearized vector. According to the following system (0.5-4μL of double-stranded DNA fragment, 0.5-4μL of linearized vector, 2μL of T4 ligase, XμL of ddH2O, total volume 10μL), the double-stranded DNA and the linearized vector were mixed, and T4 DNA ligase ligation reaction was performed and incubated at room temperature for 30 minutes to obtain pTCRISPR-sgRNA BISG1 Knockout vector. Transformed into DH5α competent medium, selected by kanamycin, single clones were selected for colony PCR identification, plasmids were extracted from positive colonies and sequenced to confirm pTCRISPR-sgRNA BISG1 Correct, genetic transformation can be carried out.

[0070] BISG1 knockout transgenic rice (BISG1-KO1 and BISG1-KO2) was produced by Boyuan Biotechnology Company.

[0071] Example 3 Construction of BISG1 plant expression vector

[0072] Construction of BISG1 overexpression vector pRHVc-BISG1

[0073] The cDNA obtained by reverse transcription of total RNA from rice TP309 was used as template, and the cloning primers were:

[0074] F: TATCCAGATCCAGTGGGATCC ATGGCGCTGCTGGCGCGGAGGG;

[0075] R: CGCACTAGTAAGCTTGGTACC TGACAGCCTATTGAGCCCTCTG.

[0076] The PCR reaction was carried out on a PE9600 PCR instrument, and the program was 94℃ pre-denaturation for 2min; then 94℃ denaturation for 1min, 56℃ annealing for 1min, 72℃ extension for 2min, for a total of 30-32 cycles; then 72℃ extension for 10min; and 4℃ storage. The full-length cDNA of BISG1 was obtained by PCR amplification. The pRHVc-Ubi-HA-nosT vector was double-digested with BamHI and KpnI to recover the linearized vector. The above PCR recovered product BISG1 full-length cDNA and the linearized vector were mixed according to the following system (DNA fragment 0.5-5μL, linearized vector 0.5-5μL, 5×CE II Buffer 4μL, Exnase II 2μL and ddH2O XμL, total volume 20μL), and the reaction was carried out at 37℃ for 30min for homologous recombination to obtain the pRHVc-BISG1 recombinant vector.

[0077] After transformation to DH5α competent state and selection by kanamycin, single clones were picked for colony PCR identification. After plasmids were extracted from positive colonies and sequenced correctly, it was confirmed that pRHVc-BISG1 was correct and genetic transformation could be carried out.

[0078] BISG1 overexpression transgenic rice (BISG1-OE1 and BISG1-OE2) was produced by Boyuan Biotechnology Company.

[0079] Example 4 BISG1 is involved in regulating plant disease resistance

[0080] In plants, BISG1 is mainly focused on plant phosphorus starvation and drought stress. The present invention found that BISG1 plays an important role in regulating plant disease resistance. Therefore, the effect of gene BISG1 on rice disease resistance was tested.

[0081] Wounding and inoculation treatment at the seedling stage: The test materials were Taipei 309 (TP309), BISG1 knockout strains BISG1-KO1 and BISG1-KO2 with TP309 as the background and genetically stable, and BISG1 overexpression strains BISG1-OE1 and BISG1-OE2 with TP309 as the background and genetically stable. Select seeds with full grains, put them in a conical bottle filled with tap water, and place them in a dark incubator at 37°C for germination, and change the water every day. After 2 days, select white seeds and put them in a 96-well seedling plate. Put the 96-well seedling plate on a float and grow it in Hoagland nutrient solution. After 21 days, select the second to last rice leaf with the same growth and size, and inoculate 5μL of a concentration of 5×10 5 ml -1 The spores of Zhong10-8-14 rice blast fungus were inoculated and the length of the lesions was observed and counted after 5 days. The statistical results showed that after puncture inoculation, the length of the lesions of BISG1-KO1 and BISG1-KO2 plants under the TP309 background was significantly reduced and the disease resistance was enhanced compared with TP309 ( Figure 2 Compared with TP309, the lesion length of BISG1-OE1 and BISG1-OE2 plants increased significantly under the TP309 background, and the disease resistance of the plants decreased significantly ( Figure 3 ). This indicates that BISG1 negatively regulates plant disease resistance, and its encoding gene BISG1 can be used as a target gene for molecular breeding to improve plant disease resistance.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a knockout vector of a rice E3 ubiquitin ligase BISG1 encoding gene in cultivating transgenic rice resistant to rice blast, characterized in that: The amino acid sequence of the rice E3 ubiquitin ligase BISG1 is shown in SEQ ID No.2; the nucleotide sequence of the encoding gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The knockout vector of the encoding gene is constructed by using the CRISPR / Cas9 method, and the knockout vector is transferred into a rice receptor material to improve the resistance of rice to rice blast.

3. A method for improving rice blast resistance, characterized in that: The method comprises the step of down-regulating the expression level of a gene encoding rice E3 ubiquitin ligase BISG1; the nucleotide sequence of the gene encoding rice E3 ubiquitin ligase BISG1 is shown in SEQ ID No.

1.

4. The method according to claim 3, characterized in that The knockout vector of the rice E3 ubiquitin ligase BISG1 encoding gene is constructed by using the CRISPR / Cas9 method, and the knockout vector is transferred into a rice receptor material to improve the rice resistance to rice blast.

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