Application of rice circular RNA (Ribonucleic Acid) and encoding protein WRKY9-88aa thereof in aspect of improving plant disease resistance

Abstract

The invention discloses application of rice circular RNA (Ribonucleic Acid) and an encoding protein WRKY9-88aa thereof in the aspect of improving the disease resistance of plants. The invention discloses a rice circRNA (Ribonucleic Acid), the nucleotide sequence of which is shown as SEQ ID NO.1, a rice WRKY9-88aa gene is obtained by further research, and the condition that the disease resistance of plants can be improved by the protein WRKY9-88aa coded by the rice circRNA is found. Experiments prove that overexpression of the WRKY9-88aa gene can improve the disease resistance of plants, and especially the disease resistance to rice stripe mosaic disease, rice bacterial leaf blight and rice blast is remarkably improved. Besides, WRKY9-88aa is taken as a target spot, germplasm resources with enhanced disease resistance can be cultivated, great significance is achieved for cultivation of disease-resistant rice varieties, meanwhile, a new and brand-new disease-resistant strategy research direction based on circular RNA is provided, and good application prospects and value are achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, relates to the application of rice circular RNA and its encoding protein WRKY9-88aa in improving plant disease resistance. Background Art

[0002] Circular RNA (circRNA) is a type of covalently closed single-stranded RNA without 5' and 3' ends that was discovered in recent years and widely exists in eukaryotes. Due to its low expression abundance, it was once considered to be a byproduct of abnormal RNA splicing. CircRNA was first discovered in the model plant Arabidopsis in 2014, and has been reported in monocotyledonous plants such as rice, wheat, and corn, and dicotyledonous plants such as kiwi, Arabidopsis, potato, cotton, pepper, and pear.

[0003] At present, plant circRNAs mainly include the following types: (1) exon-derived circRNAs: spliced ​​from the same linear transcript, and can be formed by the circularization of one or more exons; (2) intron-derived circRNAs: formed by the circularization of introns in linear transcripts; (3) exon-intron-derived circRNAs: formed by exons and introns retained between exons; (4) intergenic region circRNAs: formed by intergenic regions separated from introns or exons by more than 1kb on linear transcripts; (5) antisense chain circRNAs: formed by one or more exons of the antisense chain of linear transcripts. In addition, exon-derived circRNAs are mainly present in the cytoplasm and can act as miRNA sponges or be translated into proteins; circRNAs containing intron sequences, such as intron-derived circRNAs and exon-intron-formed circRNAs, are mainly located in the nucleus and regulate their parental genes.

[0004] At present, circRNA has diverse functions, mainly including: (1) as miRNA sponge: bioinformatics analysis found that a large number of circRNAs contain rich miRNA binding sites, which can act as miRNA sponges, weaken the binding of miRNA to target genes, and thus regulate the expression of target genes; (2) regulating the expression of parental genes: circRNA can base pair with the sequence of parental genes to form an R-loop structure, which increases the expression of corresponding alternative splicing transcripts, thereby negatively regulating the expression of its parental genes; (3) interacting with proteins: circRNA binds to proteins regulated by mRNA, changes the splicing pattern or the stability of mRNA, and thus affects protein translation; (4) coding function: some circular RNAs are translated into proteins or peptides through cap-independent mechanisms, including internal ribosome entry sites and N6-methyladenosine modification.

[0005] Peptides encoded by circular RNA play an important regulatory role in plant development and immunity. For example, circMAP3K4 is translated to produce a 455-amino acid protein circMAP3K4-455aa, which can inhibit liver cancer cell-induced apoptosis by interacting with the apoptosis-inducing factor mitochondria-associated factor (AIF), thereby protecting AIF from cleavage, reducing its nuclear distribution, preventing hepatocellular carcinoma (HCC) cells from dying under stress, and promoting HCC development. Other studies have shown that ZNF609-250aa, a 250-amino acid functional protein encoded by circ-ZNF609, depends on the AKT / mTOR signaling pathway to induce autophagic flow disorders in renal tubular epithelial cells, promote cell apoptosis, and cause ischemic acute kidney injury (AKI). The results of this study suggest that targeting circ-ZNF609 may be a new method for the treatment of ischemic AKI. In addition, recent research reports have found that long noncoding RNA-H19 (Lnc-H19) encodes an immune-related protein-H19-IRP. The H19-IRP protein directly activates the transcription of CCL2 and Galectin-9, promotes the recruitment of myeloid-derived suppressor cells (MDSC) / tumor-associated macrophages (TAM) to produce an immunosuppressive glioblastoma tumor microenvironment (GBM-TME), indicating that the protein encoded by lncRNA has the prospect of being a target for tumor immunotherapy.

[0006] Proteins encoded by circular RNA have broad application prospects in human medicine and animal treatment research, but there is no clear research in plant research. Therefore, it is of great significance to study and find proteins encoded by circular RNA that improve plant disease resistance and apply them to resistance breeding. Summary of the invention

[0007] The present invention aims to provide a circRNA, the WRKY9-88aa protein encoded by the circRNA can improve the disease resistance of plants and has good application prospects and value in disease-resistant breeding.

[0008] The first object of the present invention is to provide a circRNA.

[0009] The second object of the present invention is to provide a WRKY9-88aa gene.

[0010] The third object of the present invention is to provide a WRKY9-88aa protein encoded by the above gene.

[0011] The fourth object of the present invention is to provide the application of the above-mentioned circRNA, gene or protein.

[0012] The fifth object of the present invention is to provide the use of the reagent for promoting the expression of the above-mentioned protein or gene.

[0013] A sixth object of the present invention is to provide a method for constructing a plant with enhanced disease resistance.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] The present invention provides a circRNA, the nucleotide sequence of the circRNA is shown as SEQ ID NO.1.

[0016] The present invention provides a WRKY9-88aa gene, the nucleotide sequence of which is a nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO.2.

[0017] As one of the specific solutions, the nucleotide sequence of the WRKY9-88aa gene is shown in SEQ ID NO.3.

[0018] The present invention provides a WRKY9-88aa protein encoded by the WRKY9-88aa gene, and the amino acid sequence thereof is shown in SEQ ID NO.2.

[0019] Application of the above circRNA, the above gene or the above protein in improving plant disease resistance.

[0020] Use of the above circRNA, the above gene or the above protein in the preparation of a reagent for improving plant disease resistance.

[0021] As an alternative embodiment, the reagent includes a gene expression cassette, an overexpression vector, an engineered bacterium or a transgenic plant cell line.

[0022] As an optional embodiment, the method for preparing the overexpression vector is: connecting the above-mentioned WRKY9-88aa gene to an expression vector.

[0023] As an alternative embodiment, the vector is a pRHV vector.

[0024] As an optional embodiment, the overexpression vector is connected to the WRKY9-88aa gene and the expression vector by homologous recombination, wherein the restriction endonucleases are BamHI and HindIII.

[0025] As an alternative embodiment, the homologous recombination primers of WRKY9-88aa gene include:

[0026] pRHV-WRKY9-88aa-F:gatccagtgggatccATGGGTGCCAATGGAGGAAG;

[0027] pRHV-WRKY9-88aa-R: cgcacgagtaagcttTCAGCGAAGGGGACGACGC.

[0028] As an optional embodiment, the method for preparing the engineered bacteria is: transforming the above-mentioned overexpression vector into Agrobacterium.

[0029] The use of the above circRNA, the above gene or the above protein as a target in improving plant disease resistance.

[0030] Specifically, the plant is rice.

[0031] Specifically, the disease resistance refers to resistance to the following diseases: one or more of diseases caused by rice stripe mosaic virus, diseases caused by rice bacterial blight pathogenic bacteria, and diseases caused by rice blast fungus.

[0032] The use of the reagent for promoting the expression of the above gene or the above protein in preventing and controlling plant diseases, wherein the plant diseases include one or more of rice stripe mosaic disease, rice bacterial leaf blight and rice blast.

[0033] Use of an agent for promoting the expression of the above gene or protein in the preparation of a product for preventing and controlling plant diseases, wherein the plant diseases include one or more of rice stripe mosaic disease, rice bacterial leaf blight, and rice blast.

[0034] The present invention provides a method for constructing a plant with enhanced disease resistance, which comprises overexpressing the WRKY9-88aa gene in the plant.

[0035] As an optional embodiment, the method for constructing a plant with enhanced disease resistance is: constructing a WRKY9-88aa gene overexpression vector, transferring the WRKY9-88aa gene overexpression vector into Agrobacterium, and then using Agrobacterium to infect the plant.

[0036] The present invention has the following beneficial effects:

[0037] The present invention obtained a rice circRNA and found that the protein WRKY9-88aa encoded by it can improve the disease resistance of plants. Experiments have shown that overexpression of the WRKY9-88aa gene can improve the disease resistance of plants, especially the resistance to rice stripe mosaic disease, rice bacterial blight and rice blast, which has been significantly improved, proving that WRKY9-88aa is a key disease-resistant gene in plants. In addition, the present invention uses WRKY9-88aa as a target to cultivate germplasm resources with enhanced disease resistance, which is of great significance for the cultivation of disease-resistant rice varieties. At the same time, it provides a new research direction for disease resistance strategies based on circular RNA, which has good application prospects and value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Figure 4 shows the disease symptoms of rice overexpressing WRKY9-88aa 45 days after inoculation with RSMV. WT represents the wild-type rice variety ZH11.

[0039] Figure 2 Analysis of viral nucleoprotein accumulation 45 days after RSMV infection of ZH11 and WRKY9-88aa rice.

[0040] Figure 3 Figure 2 shows the symptoms of rice overexpressing WRKY9-88aa 20 days after infection with rice blast disease. WT represents the wild-type rice variety ZH11.

[0041] Figure 4 This is a statistical analysis of the lesion area on rice leaves 20 days after infection with rice blast.

[0042] Figure 5 Figure 1 shows the symptoms of rice overexpressing WRKY9-88aa 14 days after infection with bacterial blight. WT represents the wild-type rice variety ZH11.

[0043] Figure 6 The data are statistics of the lesion lengths on rice leaves 14 days after infection with bacterial blight. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0045] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0046] In the following examples, the competent E. coli DH5α was purchased from Shanghai Weidi Biotechnology Co., Ltd. with the product number DL1001S.

[0047] Gel recovery kit, brand is Axygen, product number is AP-GX-50.

[0048] IIOne Step Cloning Kit, brand is Vazyme, item number is C112-01.

[0049] Plasmid extraction kit, brand is Axygen, product number is MAG-PS.

[0050] The pRHV vector has been disclosed in the document "F. He, F. Zhang, WX Sun, YS Ning, GL Wang, Aversatile vector toolkit for functional analysis of rice genes. Rice 11, 27 (2018)".

[0051] Rice stripe mosaic virus (RSMV) has been disclosed in the document "X. Yang, et al., Rice stripe mosaic virus, a novel cytorhabdovirus infecting rice via leafhopper transmission. Front. Microbiol. 7, 2140 (2016)".

[0052] The bacterial blight pathogen (Xoo PXO99A strain) and the rice blast pathogen (M.oryzae EA18) were donated and preserved by the laboratories of Professor Cui Zining and Professor Sha Gan (South China Agricultural University), respectively, and have been published in the document "G.Sha, et al., Genomeediting of a rice CDP-DAG synthase confers multipathogen resistance. Nature 618, 1017-1023 (2023)".

[0053] The rice variety in the following examples is Zhonghua 11 (ZH11), and the seeds are propagated and preserved in this laboratory.

[0054] The nucleotide sequence of WRKY9-88aa in the present invention is shown as SEQ ID No. 2 in the sequence table.

[0055] The present invention uses rice overexpressing WRKY9-88aa as a research object and wild-type rice ZH11 as a control.

[0056] Example 1 Construction and identification of WRKY9-88aa overexpression vector

[0057] a) Obtaining the target gene WRKY9-88aa

[0058] The present invention has obtained a circRNA, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0059] SEQ ID NO.1:

[0060] ACACACCGGACCGCGGCGACGACGACGGCGACGACGCAGCAATTCCTGGAGCCGCGAGCGTCGTCCACGGCGCAGGCCACCGCCGACGCGGACATGGCAGCGTCCGACGACGAGGCCGGCCGCGGCGGCGGCGACGGCGACGCGTCGTCCCCTTCGCTGAGCAACGCCGCCGGCGGCGGCGGCGGCGGCAACAAGATGAGGAGGGTTGGTGGTCAAGACGAGACTGCGGCGGCGGCGCCGGCGAGGGAGAACGGCGAGCAGCAGGCGGCGGCGGCGGCCGAGCTGCCGTGCCGGAAACCGAGGGTGTCCGTGCGTGCGCGATCCGAAGCACCAATGATTAGTGATGGGTGCCAATGGAGGAAGTACGGGCAGAAAATGGCAAAGGGGAATCCATGCCCGCGAGCGTACTACCGATGCACAATGGCTATCGGATGCCCAGTCAGGAAGCAG。

[0061] The amino acid sequence of the WRKY9-88aa protein encoded by circRNA is shown in SEQ ID NO.2, SEQ ID NO.2:

[0062] MGANGGSTGRKWQRGIHARERTTDAQWLSDAQSGSRHTGPRRRRRRRR SNSWSRERRPRRRPPPTRTWQRPTTRPAAAAATATRRPLR。

[0063] The nucleotide sequence of WRKY9-88aa is shown in SEQ ID NO.3,

[0064] SEQ ID NO.3:

[0065] ATGGGTGCCAATGGAGGAAGTACGGGCAGAAAATGGCAAAGGGGAATCCATGCCCGCGAGCGTACTACCGATGCACAATGGCTATCGGATGCCCAGTCAGGAAGCAGACACACCGGACCGCGGCGACGACGACG GCGACGACGCAGCAATTCCTGGAGCCGCGAGCGTCGTCCACGGCGCAGGCCACCGCCGACGCGGACATGGCAGCGTCCGACGACGAGGCCGGCCGCGGCGGCGGCGACGGCGACGCGTCGTCCCCTTCGCTGA.

[0066] Based on the WRKY9-88aa nucleic acid sequence, the sequence primers (pRHV-WRKY9-88aa-F / R) for amplifying the WRKY9-88aa gene were designed using the plasmid containing the WRKY9-88aa gene as a template. The PCR amplification products were separated by agarose gel electrophoresis, and the target gene fragment was recovered using a gel recovery kit (Axygen). The specific steps were carried out according to the kit instructions.

[0067] The specific primer sequences of pRHV-WRKY9-88aa-F / R are as follows (5'-3'):

[0068] pRHV-WRKY9-88aa-F:gatccagtgggatccATGGGTGCCAATGGAGGAAG,

[0069] pRHV-WRKY9-88aa-R: cgcacgagtaagcttTCAGCGAAGGGGACGACGC.

[0070] In the primer sequence of pRHV-WRKY9-88aa-F / R, lowercase letters represent homologous sequences of the vector, italic lowercase letters represent BamHⅠ and HindⅢ restriction sites, and uppercase letters represent specific sequences of the WRKY9-88aa gene.

[0071] b) Enzyme-digested linearized vector

[0072] The pRHV empty vector plasmid was subjected to double restriction digestion experiment using two restriction endonucleases, BamHI and HindⅢ. The vector digestion product was recovered using a gel recovery kit (Axygen). The specific steps were carried out according to the kit instructions.

[0073] c) Homologous recombination

[0074] use IIOne Step Cloning Kit (Vazyme), operate according to the instructions of the kit, connect the target gene fragment and the vector enzyme digestion product by homologous recombination, the specific method is as follows:

[0075] Reaction system: 5×CEⅡBuffer 2μL, gel recovery product 2μL, plasmid enzyme digestion product 1μL, Ⅱ 1μL and ddH2O 4μL, the total system is 10μL.

[0076] Reaction procedure: 37°C, 30 min; store at 4°C or directly transform E. coli.

[0077] d) Transformation of recombinant plasmid into E. coli

[0078] The homologous recombination product was transformed into Escherichia coli competent DH5α (Shanghai Weidi Biotechnology Co., Ltd.), and the transformation method was carried out according to the instructions of the competent product.

[0079] e) Screening and identification of positive transformants and extraction of recombinant plasmids

[0080] Pick the monoclonal colonies on the plate for colony PCR, and confirm the positive transformants after agarose gel electrophoresis. In the clean bench, inoculate the colonies identified as positive clones into 5mL of liquid LB medium containing the corresponding antibiotics, place them on a shaker at 37°C, and shake and culture at 200rpm for 8-12h. After preserving the glycerol bacteria, extract the plasmid and send it to the company for sequencing identification. The plasmids that have been confirmed to be correct in the sequencing results are extracted using Axygen's plasmid extraction kit according to its instructions.

[0081] The extracted part of the plasmids was handed over to BioGene Technology (Jiangsu) Co., Ltd. for genetic transformation of rice. The rice variety ZH11 was used as the transformation background to obtain transgenic rice plants overexpressing the WRKY9-88aa gene.

[0082] Example 2 Disease resistance analysis of transgenic rice overexpressing WRKY9-88aa

[0083] 1. Rice seed germination and cultivation

[0084] Rice seeds of ZH11 variety (control) and seeds of two transgenic rice plants overexpressing WRKY9-88aa gene (plant numbers #3 and #12, respectively) were selected, and the rice seeds were placed in a 37°C incubator and soaked in clean water for one day, then wrapped with moist gauze for germination, and after the seeds turned white, they were evenly sprinkled on moist culture soil, and a layer of matrix soil was sprinkled on the surface. The germination was completed after culturing for 2 days. The culture conditions were set at 28°C, 80% humidity, and a light cycle of light period (L): dark period (D) = 16:8.

[0085] 2. Rice Stripe Mosaic Virus (RSMV) Resistance Assay

[0086] The rice seeds that have completed germination were placed in a small black square pot and cultured until the three-leaf stage (culture conditions were set at 28°C, humidity 80%, and the photoperiod was light period (L): dark period (D) = 16:8). At the same time, 1-2-year-old electric leafhopper nymphs were placed on RSMV-infected rice to feed. After 14 days, the electric leafhoppers were placed in a small black square pot covered with a perforated plastic bag, and the rice seedlings at the 3-4 leaf stage were infected with the virus. The inoculation intensity was 1:2 (the ratio of the number of rice plants to be inoculated to the number of poisonous insects), and the insects were driven away every 10 hours. After 2 days, the electric leafhoppers were removed, and the rice inoculated with RSMV was transplanted to the experimental field for continued cultivation. Samples were taken after 14 days, and qPCR detection was performed using the internal reference gene EF1a primer and RSMV quantitative primers, and the positive strains were retained for subsequent experiments.

[0087] The symptoms of rice after infection were observed 45 days after infection, and total RNA was extracted from rice leaves. The protein content of RSMV was detected by Western blot.

[0088] The specific sequences (5'-3') of the primers used for qPCR are:

[0089] Primer F for the internal reference gene EF1a: ACATTGCCGTCAAGTTTGCTG;

[0090] Primer R for the internal reference gene EF1a: AACAGCCACCGTTTGCCTC;

[0091] Quantitative primer F for RSMV: TCTGGGTGAAGTGTCTTCTCA;

[0092] The quantitative primer R of RSMV is: GCCCTCTTCTAATGTGCGG.

[0093] The results showed that compared with the wild-type ZH11 (WT), the symptoms of leaf shrinkage and mosaic in rice overexpressing WRKY9-88aa were significantly alleviated ( Figure 1 ), viral protein accumulation was significantly reduced ( Figure 2 ), indicating that overexpression of WRKY9-88aa inhibited RSMV infection.

[0094] 3. Determination of resistance to rice blast

[0095] The rice seeds that have been germinated are placed in a small black square pot and cultured until the three-leaf stage, and then transplanted to a blue plastic box and cultured until the early tillering stage. The culture medium of the cultured bacteria is separated into equal-sized bacterial blocks, and a hole is punched 5 cm away from the tip of the rice leaf to create a wound. The bacterial block is placed on the wound and fixed with transparent tape. The area of ​​the lesion is observed and counted after 20 days.

[0096] The results showed that compared with the wild-type ZH11 (WT), the brown spot area of ​​the inoculated leaves of rice overexpressing WRKY9-88aa was significantly reduced ( Figure 3 and Figure 4 ), indicating that overexpression of WRKY9-88aa inhibited the infection of rice blast fungus.

[0097] 4. Resistance determination to rice bacterial blight

[0098] Place the germinated rice seeds in a small black square pot and culture them until the three-leaf stage, then transplant them to a blue plastic box and culture them until the early tillering stage. Take 100 μL of the preserved bacterial strain of white leaf blight and add it to 5 mL of M210 medium, transfer it to 28°C, shake it on a shaker at 200 rpm for 36-48 hours, add 100 μL of the activated strain to 30 mL of M210 medium, and shake it under the same conditions until the OD value is 1.0; use sterilized scissors to dip into the bacterial solution and cut the rice leaves vertically 2 cm from the tip for inoculation; measure the diseased length of the leaves 14 days after inoculation.

[0099] The results showed that compared with wild-type ZH11 (WT), the leaf wilting symptoms of WRKY9-88aa overexpressing rice were significantly alleviated ( Figure 5 and Figure 6 ), indicating that overexpression of WRKY9-88aa inhibited the infection of Xanthomonas oryzae.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A circRNA, characterized in that: The nucleotide sequence of the circRNA is shown in SEQ ID NO.

1.

2. A WRKY9-88aa gene, characterized in that: The nucleotide sequence thereof is a nucleotide sequence capable of encoding the amino acid sequence shown in SEQ ID NO.

2.

3. A WRKY9-88aa protein encoded by the WRKY9-88aa gene according to claim 2, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

4. Use of the circRNA according to claim 1, the gene according to claim 2 or the protein according to claim 3 in improving plant disease resistance.

5. Use of the circRNA according to claim 1, the gene according to claim 2 or the protein according to claim 3 in the preparation of an agent for improving plant disease resistance.

6. Use of the circRNA according to claim 1, the gene according to claim 2 or the protein according to claim 3 as a target in improving plant disease resistance.

7. The use according to any one of claims 4 to 6, characterized in that: The disease resistance refers to resistance to the following diseases: one or more of the diseases caused by rice stripe mosaic virus, the diseases caused by rice bacterial blight pathogenic bacteria, and the diseases caused by rice blast fungus.

8. Use of an agent for promoting the expression of the gene according to claim 2 or the protein according to claim 3 in preventing and controlling plant diseases, wherein the plant diseases include one or more of rice stripe mosaic disease, rice bacterial leaf blight, and rice blast.

9. Use of an agent for promoting the expression of the gene according to claim 2 or the protein according to claim 3 in the preparation of a product for preventing and controlling plant diseases, wherein the plant diseases include one or more of rice stripe mosaic disease, rice bacterial leaf blight, and rice blast.

10. A method for constructing a plant with enhanced disease resistance, characterized in that: Overexpressing the WRKY9-88aa gene of claim 2 in a plant.

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