Application of rice circRNA and its encoded protein WRKY9-88aa in improving plant disease resistance

Abstract

The application discloses a rice circular RNA and application of a protein WRKY9-88aa coded by the rice circular RNA in improving plant disease resistance. The application discloses a rice circRNA, a nucleotide sequence of which is shown as SEQ ID NO. 1, and further research obtains a rice WRKY9-88aa gene, and it is found that the protein WRKY9-88aa coded by the gene can improve the disease resistance of plants. Experiments prove that overexpression of the WRKY9-88aa gene can improve the disease resistance of plants, especially the disease resistance to rice stripe mosaic disease, rice white leaf blight and rice blast, and the disease resistance is significantly improved. In addition, the application takes the WRKY9-88aa as a target, and can cultivate germplasm resources with enhanced disease resistance, has great significance for the cultivation of rice disease-resistant varieties, simultaneously provides a new and whole disease-resistant strategy research direction based on circular RNA, and has good application prospect and value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology. More specifically, it relates to the application of rice circular RNA and its encoded protein WRKY9-88aa in improving plant disease resistance. BACKGROUND

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

[0003] At present, plant circRNAs mainly include the following types: (1) Exon-derived circRNA: formed by the same linear transcript splicing, which can be circularized by one or more exons; (2) Intron-derived circRNA: formed by the intron of a linear transcript; (3) Exon-intron-derived circRNA: formed by exons and introns retained between exons; (4) Inter-genic region circRNA: formed by the intergenic region on the linear transcript which is separated from the intron or exon by more than 1 kb; (5) Antisense strand circRNA: formed by one or more exons of the antisense strand of a linear transcript. In addition, exon-derived circRNAs mainly exist 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-derived circRNAs, are mainly located in the nucleus and regulate their parent genes.

[0004] At present, circRNAs have diverse functions, mainly including: (1) as miRNA sponges: bioinformatics analysis has found that a large number of circRNAs contain abundant miRNA binding sites, which can act as miRNA sponges to weaken the binding of miRNA to target genes, thereby regulating the expression of target genes; (2) regulating the expression of parent genes: circRNAs can form R-loop structures by base pairing with the sequences of parent genes, increasing the expression of corresponding alternative splicing transcripts, and thereby negatively regulating the expression of parent genes; (3) interacting with proteins: circRNAs bind to mRNA-regulated proteins, changing the splicing pattern or stability of mRNA, thereby affecting protein translation; (4) encoding functions: some circular RNAs are translated into proteins or polypeptides through a cap-independent mechanism, including internal ribosome entry sites and N6-methyladenosine modification.

[0005] CircRNA-encoded peptides play an important regulatory role in plant development and immunity. For example, circMAP3K4 translation produces a 455-amino-acid protein, circMAP3K4-455aa, which can inhibit apoptosis induced by hepatocellular carcinoma cells by interacting with apoptosis-inducing factor mitochondria-associated factor (AIF), thus protecting AIF from being cleaved, reducing its nuclear distribution, preventing hepatocellular carcinoma (HCC) cells from dying under stress, and promoting HCC development. Studies have shown that ZNF609-250aa, a functional protein consisting of 250 amino acids encoded by circ-ZNF609, induces autophagy flow disorders in renal tubular epithelial cells, promotes cell apoptosis, and causes ischemic acute kidney injury (AKI), suggesting that targeting circ-ZNF609 may be a new method for treating ischemic AKI. In addition, recent research reports have found that long non-coding RNA-H19 (Lnc-H19) encodes an immune-related protein, H19-IRP, which promotes the recruitment of myeloid-derived suppressor cells (MDSCs) / tumor-associated macrophages (TAMs) by directly activating the transcription of CCL2 and Galectin-9, thus producing an immunosuppressive glioblastoma tumor microenvironment (GBM-TME), indicating that lncRNA-encoded proteins have the potential to be used as targets for tumor immunotherapy.

[0006] Based on the wide application prospects of circRNA-encoded proteins in human medicine and animal therapy research, there is no clear research in plant research. Therefore, it is of great significance to study and find circRNA-encoded proteins that can improve plant disease resistance and apply them to resistance breeding. SUMMARY

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

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

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

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

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

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

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

[0014] The above-mentioned objective of this invention is achieved through the following technical solution:

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

[0016] This 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 specific embodiment, 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 above-mentioned WRKY9-88aa gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0019] Application of the above-mentioned circRNAs, genes, or proteins in improving plant disease resistance.

[0020] The application of the above-mentioned circRNA, genes, or proteins in the preparation of reagents to enhance plant disease resistance.

[0021] As an alternative implementation, the reagents include gene expression cassettes, overexpression vectors, engineered bacteria, or transgenic plant cell lines.

[0022] As an alternative implementation, the overexpression vector is prepared by ligating the WRKY9-88aa gene to the expression vector.

[0023] As an alternative implementation, the carrier is a pRHV carrier.

[0024] As an alternative implementation, the overexpression vector connects the WRKY9-88aa gene to the expression vector via homologous recombination, with BamHⅠ and HindⅢ selected as restriction endonucleases.

[0025] As an alternative implementation method, the homologous recombination primers for the WRKY9-88aa gene include:

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

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

[0028] As an alternative implementation, the engineered bacteria are prepared by converting the above-mentioned overexpression vector into Agrobacterium.

[0029] The application of the above-mentioned circRNAs, genes, or proteins as targets in improving plant disease resistance.

[0030] Specifically, the plant in question is rice.

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

[0032] The application of reagents that promote the expression of the above-mentioned genes or proteins in the prevention and control of plant diseases, including one or more of rice stripe mosaic virus, rice bacterial blight, and rice blast.

[0033] The application of reagents that promote the expression of the above-mentioned genes or proteins in the preparation of products for the prevention and control of plant diseases, wherein the plant diseases include one or more of rice stripe mosaic virus, rice bacterial blight, and rice blast.

[0034] This invention provides a method for constructing plants with enhanced disease resistance by overexpressing the WRKY9-88aa gene in the plant.

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

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

[0037] This invention obtained a rice circRNA and discovered that its encoded protein WRKY9-88aa can enhance plant disease resistance. Experiments showed that overexpression of the WRKY9-88aa gene significantly improved plant disease resistance, particularly against rice stripe mosaic virus, rice bacterial blight, and rice blast, demonstrating that WRKY9-88aa is a key disease resistance gene in plants. Furthermore, this invention, using WRKY9-88aa as a target, can cultivate germplasm resources with enhanced disease resistance, which is of great significance for the breeding of disease-resistant rice varieties. It also provides a novel research direction for disease resistance strategies based on circular RNA, with excellent application prospects and value. Attached Figure Description

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

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

[0040] Figure 3 To overexpress the symptoms of WRKY9-88aa rice 20 days after infection with rice blast, WT represents the wild-type rice variety ZH11.

[0041] Figure 4 Statistics on the area of ​​lesions on rice leaves 20 days after infection with rice blast.

[0042] Figure 5 To overexpress the symptoms of bacterial blight in WRKY9-88aa rice 14 days after infection, WT represents the wild-type rice variety ZH11.

[0043] Figure 6 Statistics on the length of lesions on rice leaves 14 days after infection with bacterial blight. Detailed Implementation

[0044] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0046] In the following examples, Escherichia coli competent cells DH5α were purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number DL1001S.

[0047] The gel recovery kit is from Axygen and its catalog number is AP-GX-50.

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

[0049] The plasmid extraction kit is from Axygen and its catalog number is MAG-PS.

[0050] The pRHV vector has been published in the paper “F.He,F.Zhang,WXSun,YSNing,GLWang,Aversatilevector toolkit for functional analysis of rice genes.Rice 11,27(2018)”.

[0051] Rice stripe mosaic virus (RSMV) has been published in the literature 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 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 literature "G. Sha, et al., Genome editing of a rice CDP-DAG synthase confers multipathogen resistance. Nature 618, 1017-1023 (2023)".

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

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

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

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

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

[0058] This invention has yielded 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, primers (pRHV-WRKY9-88aa-F / R) for amplifying the WRKY9-88aa gene were designed using a 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 performed according to the kit instructions.

[0067] The specific primer sequence for pRHV-WRKY9-88aa-F / R is as follows (5'-3'):

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

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

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

[0071] b) Enzyme digestion of linearized vector

[0072] The pRHV empty vector plasmid was double-digested using two restriction endonucleases, BamHI and HindIII. The digested products were recovered using a gel recovery kit (Axygen). The specific steps were performed according to the kit instructions.

[0073] c) Homologous recombination

[0074] use The IIOne Step Cloning Kit (Vazyme) was used in accordance with the kit's instructions to ligate the target gene fragment with the vector digestion product via homologous recombination. The specific method is as follows:

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

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

[0077] d) Transformation of Escherichia coli with recombinant plasmids

[0078] The homologous recombination product was transformed into competent E. coli DH5α cells (Shanghai Weidi Biotechnology Co., Ltd.), and the transformation method was carried out in accordance with the product instructions for competent cells.

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

[0080] Single colonies were picked from the plates for colony PCR, and positive transformants were confirmed by agarose gel electrophoresis. In a clean bench, colonies confirmed as positive clones were inoculated into 5 mL of liquid LB medium containing the appropriate antibiotic and incubated at 37°C with shaking at 200 rpm for 8–12 h. After glycerol preservation, plasmids were extracted and sent to the company for sequencing identification. Plasmids with confirmed sequencing results were extracted using Axygen's plasmid extraction kit according to its instructions.

[0081] A portion of the extracted plasmids was given to Baige Gene Technology (Jiangsu) Co., Ltd. for genetic transformation of rice. Using the rice variety ZH11 as the transformation background, transgenic rice plants overexpressing the WRKY9-88aa gene were obtained.

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

[0083] I. Rice Seed Germination and Cultivation

[0084] Select rice seeds of variety ZH11 (control) and seeds from two transgenic rice plants overexpressing the WRKY9-88aa gene (plant numbers #3 and #12, respectively). Soak the rice seeds in water at 37℃ for one day, then wrap them in moist gauze to promote germination. After the seeds show white sprouts, evenly scatter them on moist culture soil and cover the surface with a layer of substrate soil. Germination is completed after 2 days of cultivation. The cultivation conditions are set at 28℃, 80% humidity, and a photoperiod of light period (L): dark period (D) = 16:8.

[0085] II. Rice Stripe Mosaic Virus (RSMV) Resistance Determination

[0086] Germinated rice seeds were cultured in small black square pots until the three-leaf stage (culture conditions set at 28℃, 80% humidity, and a photoperiod of light period (L):dark period (D) = 16:8). Simultaneously, 1st-2nd instar nymphs of the electric leafhopper were placed on RSMV-infected rice plants to feed. After 14 days, the electric leafhoppers were placed in small black square pots covered with perforated plastic bags to transmit the virus to 3-4 leaf stage rice seedlings at a virus inoculation ratio of 1:2 (ratio of rice plants to insects). Insects were removed approximately every 10 hours. Two days later, the electric leafhoppers were removed, and the RSMV-infected rice was transplanted to the experimental field for further cultivation. Samples were taken 14 days later, and qPCR detection was performed using primers for the internal control gene EF1a and RSMV quantitative primers. Positive strains were retained for subsequent experiments.

[0087] The disease symptoms of rice 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 as follows:

[0089] Internal reference gene EF1a primer F: ACATTGCCGTCAAGTTTGCTG;

[0090] Internal reference gene EF1a primer R: AACAGCCACCGTTTGCCTC;

[0091] RSMV quantitative primer F: TCTGGGTGAAGTGTCTTCTCA;

[0092] The quantitative primer R for RSMV is: GCCTCTTCTAATGTGCGG.

[0093] The results showed that compared with wild-type ZH11 (WT), rice overexpressing WRKY9-88aa exhibited significantly reduced symptoms such as leaf wrinkling and mosaic patterns. Figure 1 The accumulation of viral proteins was significantly reduced. Figure 2 This indicates that overexpression of WRKY9-88aa inhibited RSMV infection.

[0094] III. Rice Blast Resistance Test

[0095] Germinated rice seeds were placed in small black square pots and cultured until the three-leaf stage, then transplanted to blue plastic square frames and cultured until the early tillering stage. The culture medium for the inoculum was separated into equal-sized mycelial blocks. A hole was made 5 cm away from the tip of the rice leaf to create a wound. The mycelial blocks were placed in the wound and fixed with transparent tape. The area of ​​the lesions was observed and counted after 20 days.

[0096] The results showed that compared with wild-type ZH11 (WT), the brown spot symptom at the inoculation site of rice overexpressing WRKY9-88aa was significantly reduced. Figure 3 and Figure 4 The results indicate that overexpression of WRKY9-88aa inhibited the infection of rice blast fungus.

[0097] IV. Resistance test for rice bacterial leaf blight

[0098] Germinated rice seeds were cultured in small black square pots until the three-leaf stage, then transplanted to blue plastic frames and cultured until the early tillering stage. 100 μL of the preserved *Bacillus thuringiensis* strain was added to 5 mL of M210 medium and cultured at 28°C with shaking at 200 rpm for 36-48 hours. 100 μL of the activated strain was added to 30 mL of M210 medium and cultured under the same conditions until the OD value reached 1.0. Using sterilized scissors dipped in the bacterial solution, rice leaves were vertically cut 2 cm from the leaf tip for inoculation. The length of diseased leaves was measured 14 days after inoculation.

[0099] The results showed that compared with wild-type ZH11 (WT), rice overexpressing WRKY9-88aa exhibited significantly reduced leaf wilting symptoms. Figure 5 and Figure 6 The results indicate that overexpression of WRKY9-88aa inhibited the infection of rice bacterial blight pathogen.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within 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 kind WRKY9-88aa Genes, characterized by, Its nucleotide sequence is a nucleotide sequence that can encode the amino acid sequence shown in SEQ ID NO.

2.

3. A device as described in claim 2 WRKY9-88aa The WRKY9-88aa protein encoded by the gene is characterized by, Its amino acid sequence is shown in SEQ ID NO.

2.

4. The application of the circRNA of claim 1, the gene of claim 2, or the protein of claim 3 in improving plant disease resistance, characterized in that, The plant is rice, and the disease resistance refers to resistance to one or more of the following diseases: diseases caused by rice stripe mosaic virus, diseases caused by rice bacterial blight pathogens, and diseases caused by rice blast fungus.

5. The use of the circRNA of claim 1, the gene of claim 2, or the protein of claim 3 in the preparation of reagents to enhance plant disease resistance, characterized in that, The plant is rice, and the disease resistance refers to resistance to one or more of the following diseases: diseases caused by rice stripe mosaic virus, diseases caused by rice bacterial blight pathogens, and diseases caused by rice blast fungus.

6. The application of the reagent for promoting the expression of the gene according to claim 2 or the protein according to claim 3 in the prevention and control of plant diseases, characterized in that, The reagent is a vector for overexpressing the gene of claim 2, and the plant disease is one or more of the following: a disease caused by rice stripe mosaic virus, a disease caused by rice bacterial blight pathogen, and a disease caused by rice blast fungus.

7. The use of the reagent for promoting the expression of the gene according to claim 2 or the protein according to claim 3 in the preparation of products for the prevention and control of plant diseases, characterized in that, The reagent is a vector for overexpressing the gene of claim 2, and the plant disease is one or more of the following: a disease caused by rice stripe mosaic virus, a disease caused by rice bacterial blight pathogen, and a disease caused by rice blast fungus.

8. A method for constructing plants with enhanced disease resistance, characterized in that, Overexpression of the substance described in claim 2 in plants WRKY9-88aa Gene; the plant is rice, and the disease resistance refers to resistance to one or more of the following diseases: diseases caused by rice stripe mosaic virus, diseases caused by rice bacterial blight pathogen, and diseases caused by rice blast fungus.

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