Application of gene LOC_Os06g02960 in improving resistance to rice bacterial leaf streak disease

Abstract

The present invention discloses the application of gene LOC_Os06g02960 in improving resistance to rice bacterial leaf streak, and belongs to the technical field of plant genetic engineering. The expression level of the gene is reduced after being induced by the pathogen of rice bacterial leaf streak in susceptible haploid materials. After knocking out the gene, resistance to rice bacterial leaf streak can be improved. The LOC_Os06g02960 editing site sequence is connected to the gene editing vector pRGEB32S to transform rice, and the results show that the gene can negatively regulate resistance to rice bacterial leaf streak. The gene editing vector constructed by the present invention can be used in rice breeding for resistance to bacterial leaf streak. The present invention helps to better understand the mechanism of action of LOC_Os06g02960, and the cloning of LOC_Os06g02960 lays the foundation for further understanding the interaction between rice bacterial leaf streak pathogens and the disease resistance signal transduction pathway, and has great application value in breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to gene LOC_Os06g02960 Application in improving resistance to bacterial leaf streak disease in rice. Background Art

[0002] Bacterial leaf streak (BLS) is caused by Xanthomonas oryzae pv. Oryzicola ( XO ) is a bacterial disease caused by BLS. The disease causes 8%–32% loss in rice yield. The simplest way to prevent BLS is to use chemical pesticides, but the large-scale use of chemical pesticides not only pollutes the environment but also poses food safety issues. In addition, XO It mainly infects rice leaves through stomata or wounds. Once it encounters severe weather conditions such as typhoons and forms a large-scale infection, it is difficult to effectively control it with pesticides. With the continuous change of climate, XO The degree of damage caused by the disease is also increasing year by year. Therefore, cultivating and improving disease-resistant rice varieties is the most effective way to prevent and control the disease and ensure food security.

[0003] At present, most studies on BLS resistance have focused on biparental genetic mapping populations. At least 13 BLS resistance QTLs have been identified through classical QTL mapping methods, but only QUR and bls1 Key genes in x5 and OsMAPK6 Successfully cloned; at least 22 BLS resistance QTLs were identified through genome-wide association studies (GWAS), but the functional genes are not well understood. R Gene-- RxO1 , which is resistant to all African strains tested, but ineffective against many Asian strains. In addition, 22 genes have been reported to be associated with BLS resistance in rice. In order to cope with the increasing severity of BLS, it is particularly important to identify new disease resistance genes / QTLs.

[0004] Large-scale identification of germplasm resistance helps to identify more resistance genes / QTLs. Aggregating multiple resistance genes / QTLs through marker-assisted selection is very important for improving the broad-spectrum and durable resistance of rice. With the reduction of sequencing costs, GWAS based on whole genome resequencing has been widely used in the field of plant disease resistance research. Due to the influence of marker density and linkage disequilibrium (LD), GWAS cannot provide accurate target genes at a given locus. Transcriptome analysis can overcome this limitation by detecting and distinguishing the expression of candidate genes in different genotypes.

[0005] Identification of disease resistance-related genes helps to reveal the disease resistance mechanism of rice and the specific interaction mechanism between rice and pathogens. Further selection of disease-resistant varieties can better control and reduce the damage of rice bacterial leaf streak to rice and enhance the disease resistance of plants. These studies have important application value for rice gene function research and disease-resistant rice breeding. Summary of the invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a LOC_Os06g02960 Application in improving resistance to bacterial leaf streak disease in rice.

[0007] The present invention relates to the identification and cloning of plant resistance genes, and a new gene that negatively regulates resistance to rice bacterial leaf streak disease has been discovered LOC_Os06g02960 , the gene is located on chromosome 6, and the gene locus number is LOC_Os06g02960 (MSU accession number), its full-length genomic sequence is 2568 bp (SEQ ID NO: 1), including 5′ untranslated region (5′UTR), 1 exon (SEQ ID NO: 2) and 3′ untranslated region (3′UTR). Its cDNA is 2301 bp long (SEQ ID NO: 3) and encodes 767 amino acids. LOC_Os06g02960 The encoded protein sequence is shown in SEQ ID NO:4.

[0008] This gene is divided into two different haplotypes in the natural population of this study. The loss of this gene will increase the resistance to rice bacterial leaf streak disease. XO The infection of rice germplasm reduces the expression of this gene in susceptible germplasm. LOC_Os06g02960 The different haplotypes of rice or the CRISPR / Cas9 gene editing technology can be used to perform site-specific editing of the gene to improve the resistance of rice to bacterial leaf streak disease.

[0009] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0010] Gene LOC_Os06g02960 or its related sequences as gene editing targets, which are any one or more combinations of the following applications:

[0011] i) Application in improving resistance to bacterial leaf streak disease in rice;

[0012] ii) Application in the prevention and treatment of rice bacterial leaf streak disease;

[0013] iii) Application in breeding rice with high resistance to bacterial leaf streak disease;

[0014] ⅳ) Application in designing and screening drugs against rice bacterial leaf streak disease.

[0015] The related sequence is any one or more of the following sequences:

[0016] 1) Genes LOC_Os06g02960 The full-length nucleotide sequence of cDNA;

[0017] 2) Genes LOC_Os06g02960 The exon nucleotide sequence of

[0018] 3) Genes LOC_Os06g02960 Encoded amino acid sequence.

[0019] Furthermore, genes LOC_Os06g02960 The nucleotide sequence is shown in SEQ ID NO.1.

[0020] Furthermore, genes LOC_Os06g02960 The full-length nucleotide sequence of cDNA is shown in SEQ ID NO.2.

[0021] Furthermore, genes LOC_Os06g02960 The exon nucleotide sequence is shown in SEQ ID NO.3.

[0022] Furthermore, genes LOC_Os06g02960 The encoded amino acid sequence is shown in SEQ ID NO.4.

[0023] Furthermore, the application is gene LOC_Os06g02960 Or the use of inhibitors or blockers of its related sequences.

[0024] Furthermore, the inhibitor or blocker is used to knock out the gene LOC_Os06g02960 Gene editing vectors or their related sequences 。

[0025] Furthermore, the gene editing vector is a CRISPR / Cas9 gene editing vector.

[0026] Furthermore, the target sequence of the CRISPR / Cas9 gene editing vector is: 5'-AGCAGTACAAGTAGGAGCGG-3'.

[0027] The above genes LOC_Os06g02960 Or the use of detection primers or detection kits of related sequences in identification and / or evaluation of resistance to rice bacterial leaf streak disease.

[0028] Furthermore, the detection kit comprises at least one of the detection primers, PCR enzyme, PCR water and PCR buffer.

[0029] Furthermore, the application includes the following steps: using the detection primers or detection kit to amplify the genomic DNA or RNA of the rice variety to be detected, and judging the resistance of the target variety to rice bacterial leaf streak disease by the change of gene expression.

[0030] exist LOC_Os06g02960 A 20 bp exon containing a PAM sequence was selected as the knockout target, which was connected to the U3-gRNA expression cassette and then constructed with Cas9 into the vector pRGEB32S-LOC_Os06g02960 ， After transformation with Agrobacterium, rice callus tissue was infected. Hygromycin was used to screen knockout plants, and the results showed that the gene deletion could significantly improve resistance to rice bacterial leaf streak. The gene editing vector pRGEB32-LOC_Os06g02960 of the present invention can be used in rice breeding for resistance to bacterial leaf streak.

[0031] Mechanism of the present invention:

[0032] The inventors first identified 9 QTLs for resistance to rice bacterial leaf streak disease through genome-wide association analysis of 429 rice germplasms, including qBLS6.2 Contains the largest number of significant SNPs, peak-SNP -log 10 ( P ) value is the largest, and the peak-SNP distance LOC_Os06g02960 Recently, transcriptome sequencing has further revealed that LOC_Os06g02960 The expression level was significantly reduced 12 hours after infection with rice bacterial leaf streak. Haplotype analysis showed that haplotype 1 was more resistant than haplotype 2. Knocking out this gene in Zhonghua 11 significantly improved the resistance to rice bacterial leaf streak, and the plants became shorter. LOC_Os06g02960 The haplotype 1 of the gene is significantly shorter than the haplotype 2. The ChIP-Hub database (https: / / compbio.nju.edu.cn / app / ChIPHub / ) predicts that there is an enhancer in the exon of the gene, and there are multiple binding sites for disease resistance genes in this region, such as OsMYC2 , OsEB wait, OsMYC2 It is a transcription factor of the jasmonic acid signaling pathway. It was found through the Rice Expression Database (https: / / ricexpro.dna.affrc.go.jp / ) that this gene is mainly expressed in the stems, leaves and leaf sheaths of rice. After jasmonic acid treatment, OsMYC2 and LOC_Os06g02960 We speculate that OsMYC2 The involved jasmonic acid signaling pathway may be an important node for information regulation.

[0033] Compared with the prior art, the present invention has the following advantages and effects:

[0034] The method of the present invention uses whole genome association analysis and transcriptome sequencing analysis to identify a rice bacterial leaf streak resistance gene from rice LOC_Os06g02960 , confirmed by CRISPR / Cas9 gene editing technology LOC_ Os06g02960 It is involved in the defense response of rice to bacterial leaf streak pathogens and is an important negative regulatory gene involved in rice disease resistance. LOC_Os06g02960 The mechanism of action, LOC_Os06g02960 The identification of this gene lays a foundation for further understanding of rice-pathogen interactions and has important application value in breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the results of a survey on resistance to rice bacterial leaf streak in 429 rice germplasms.

[0036] Figure 2 It is the Manhattan plot, QQ plot and linkage disequilibrium plot of whole genome association analysis.

[0037] Figure 3 This is the Veen diagram of transcriptome sequencing and LOC_Os06g02960 Expression level diagram, R_up: up-regulated by inoculation of disease-resistant material, S_up: up-regulated by inoculation of susceptible material, R_down: down-regulated by inoculation of disease-resistant material, S_down: down-regulated by inoculation of susceptible material.

[0038] Figure 4 yes LOC_Os06g02960 Haplotype analysis diagram.

[0039] Figure 5 yes LOC_Os06g02960 Identification of deletion mutants and target site sequencing maps.

[0040] Figure 6 yes LOC_Os06g02960 Phenotype diagram of mutants, WT is Zhonghua11. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] Unless otherwise specified, the various raw materials and equipment used in the present invention are conventional commercially available products and can be directly purchased from the market.

[0043] The primer sequences used in the examples of the present invention were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0044] Example 1: Resistance survey and genome-wide association analysis of 429 rice germplasms

[0045] 1) Resistance survey of 429 rice germplasms

[0046] All germplasms were first sown in the nursery, and then the 25-day-old seedlings were transplanted to the laboratory research base. Each germplasm was planted in one row with a row spacing of 20 cm, three holes per row, and two plants per hole. Standard field management was used throughout the growing season, but no fungicide was used. The pathogen was cultured in NA medium at 28°C for 2 days and diluted with PBS solution to OD 600 =1 and add 0.5% Tween 20 as inoculant. Inoculation was carried out 45 days after rice seedlings were transplanted. Each plant was inoculated with 4 to 5 leaves with fully expanded tops. The inoculation was carried out by pressure infiltration. The bacterial solution was absorbed with a 10 ml syringe. The left hand assisted in spreading the leaves flat and blocking the injection hole of the syringe with force. Then the right hand pressed the syringe to make the bacterial liquid infiltrate into the leaves. After 21 days of inoculation, the disease was stable and a phenotypic investigation was carried out. The inoculation site was scanned with a scanner, and the lesion length was measured using Image-Pro Plus. The average lesion length after removing the extreme value was used as the resistance evaluation data. The disease levels are: immune (I): wound asymptomatic or only brown spots, high resistance (HR): lesion length 0.1-0.5 cm, resistance (R): lesion length 0.5-1.0 cm, medium resistance (MR): lesion length 1.0-1.5 cm, susceptible (S): lesion length 1.5-2.5 cm, highly susceptible (HS): lesion length greater than 2.5 cm. The results are shown in Figure 1 As shown, there are 14 highly resistant germplasms.

[0047] 2) Genome-wide association analysis

[0048] The whole genome resequencing results of 429 rice accessions were used for genotype-specific association analysis with resistance to rice bacterial leaf streak disease. The analysis software was Emmax (Kang et al., 2010). The kinship matrix of individuals was used as a covariate to correct the association results. The association threshold was 5 (based on -log 10 (1 / 335897). According to the threshold, the significantly associated SNP sites were screened, and the range of 1 LD attenuation distance upstream and downstream of the significantly associated sites was expanded as a QTL interval. Each QTL contained at least two significant SNPs. The results are shown in Figure 2 As shown in Figure 2, a total of 9 QTLs were identified, including one on chromosome 6. qBLS6.2 Has the highest peak, and the highest point is LOC_Os06g02960 Recently, using qBLS6.2The linkage disequilibrium analysis was performed on 121 SNPs with significant associations in the region, which were mainly divided into two haplotype blocks. The QQ plot showed that this model could effectively control the effects of population structure and kinship on the results of association analysis, and the association sites were more significant.

[0049] Example 2: Transcriptome sequencing analysis of disease-resistant and disease-susceptible materials

[0050] To identify differentially expressed genes induced in rice after pathogen infection, 15 resistant materials and 15 susceptible materials were selected from 429 materials. The fully expanded leaves of healthy, disease-free plants with good growth at the booting stage were inoculated with bacteria by pressure infiltration. Water inoculation was used as the control. There were 4 combinations, namely, inoculation of resistant materials, water inoculation of resistant materials, inoculation of susceptible materials, and water inoculation of susceptible materials. After 12 h, leaves 1 cm from the inoculation site were taken as samples. Leaves from each combination were mixed together as a replicate. There were 3 replicates for each combination. They were completely frozen in liquid nitrogen for 3-5 min and then transferred to an ultra-low temperature refrigerator at -80 °C for storage. RNA was extracted using an improved method. The integrity of RNA samples was checked using Nanodrop, Qubit 2.0, and Agilent 2100 bioanalyzers. cDNA library construction and sequencing were performed by Biomarker Technology Company (Beijing, China) according to standard protocols. The cDNA library products were then sequenced using an Illumina HiSeq2500 sequencing system with a read length of 125 bp. The raw sequencing reads were noise filtered using SeqPrep and Condetri_v2.0.pl to remove low-quality reads and very short sequences. The obtained high-quality reads were aligned to the Nipponbare reference genome (version IRGSP1.0; https: / / rapdb.dna.affrc.go.jp / download / irgsp1.html ). Expression profiles of mapped reads were created using Cuffdiff, and the abundance of gene expression level distributions for each gene in each sample was assessed as FPKM (fragments per kilobase of transcript per million mapped fragments). Transcripts with p-value ≤ 0.05 and log2FC ≥ 1.5 were considered differentially expressed. Functional annotation and enrichment of differentially expressed genes were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http: / / www.genome.jp / kegg / genes.html) as described by Li et al. (2015). The results are as follows Figure 3 As shown in Figure 2, a total of 3250 differentially expressed genes were identified, including LOC_Os06g02960 The expression level in the inoculated materials was lower than that in the water.

[0051] Embodiment 3: LOC_Os06g02960Haplotype analysis of

[0052] The Kidio bioinformatics cloud platform was used to obtain the variation site information of the genomic region of the target gene of all materials and the promoter region 2000 bp upstream, and all SNP sites were classified according to the method of "Genome-wide association studyusing whole-genome sequencing rapidly identifies new genesinfluencingagronomic traits in rice. Nature Genetics, 2016, 48(8):927-934". The sites with significant SNPs and non-synonymous mutations were selected for haplotype analysis, and the haplotypes with sample size less than 10 were removed. The rice germplasm phenotypic data corresponding to the haplotypes were sorted, and the box plot of the haplotypes was drawn using Origin 2021, and variance analysis was performed. The results are as follows: Figure 4 As shown, LOC_Os06g02960 The key SNPs are mainly concentrated in the exon region and can be divided into two haplotypes, and the average resistance of haplotype 1 is higher than that of haplotype 2. In the 3K database (https: / / www.rmbreeding.cn / ) LOC_Os06g02960 These five loci are mainly the two haplotypes mentioned above, and there are significant differences in plant height. The average plant height of haplotype 1 is shorter than that of haplotype 2.

[0053] Embodiment 4: LOC_Os06g02960 Construction of gene editing vector and phenotypic identification of mutants

[0054] LOC_Os06g02960 The knockout vector was constructed by converting the pRGEB32 vector (disclosed in the literature “Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. PNAS, 2015, 112(11):3570-3575”) into pRGEB32S, which was then further constructed by replacing the U3 promoter and gRNA junction sequence in pRGEB32 with a double restriction site sequence. LOC_Os06g02960The CDS sequence of the target site was searched for a suitable target site (5'-AGCAGTACAAGTAGGAGCGG-3', SEQ ID NO: 5) on the CRISPR / Cas9 online target site design website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), primers were designed according to the target site, and the synthesized primers were used for touchdown PCR to complement the target site fragment with the homology arm of pRGEB32S. The Cas9 protein expression vector pRGEB32S was used Bam HI and Kpn Ⅰ double enzyme digestion, the obtained fragment with target site was recombined with the vector, the recombinant vector was transformed into Escherichia coli DH5α and sequenced for identification, the positive transformants were used for subsequent Agrobacterium transformation experiments, and the transformation material was Zhonghua 11. Subsequently, positive plants were obtained through callus differentiation and rooting, and the knockout target of positive plants was detected by Sanger sequencing, and the expression level of transgenic materials was analyzed to further determine that the target gene was successfully knocked out. According to the mutation type, it is recorded as LOC_ Os06g02960-ko1 and LOC_Os06g02960-ko2 ( Figure 5 ). The primer sequences used are as follows:

[0055] LOC_Os06g02960 -ko-F (SEQ ID NO: 6): 5'-TGCAGATGATCCGTGGCAGGCGAGGATGAACATGACGAGTTTTAGAGCTAGAAATAG-3';

[0056] LOC_Os06g02960 -ko-R (SEQ ID NO: 7): 5'-CTATTTCTAGCTCTAAAACTCGTCATGTTCATCCTCGCCTGCCACGGATCATCTGCA-3';

[0057] Identify the correct loc_os06g02960-ko The mutants were inoculated with Psoralea oryzae at the booting stage, and the length and area of ​​leaf lesions were observed 14 days after inoculation. Figure 6 As shown, compared with the wild-type plants, loc_ os06g02960-ko The lesion length and lesion area of ​​​​the plants were relatively small, and the plant height was shorter. LOC_Os06g02960 Negative regulation of rice bacterial leaf streak resistance and plant height.

[0058] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Genes LOC_Os06g02960 The application is characterized by: Any one or more combinations of the following applications: i) Knockout of genes LOC_Os06g02960 Application in improving resistance to bacterial leaf streak disease in rice; ii) Knockout of genes LOC_Os06g02960 Application in the control of rice bacterial leaf streak disease; iii) Knockout of genes LOC_Os06g02960 Application in breeding rice with high resistance to bacterial leaf streak disease; The gene LOC_Os06g02960 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The gene according to claim 1 LOC_Os06g02960 The application is characterized by: The gene LOC_Os06g02960 The full-length nucleotide sequence of cDNA is shown in SEQ ID NO.

2.

3. The gene according to claim 1 LOC_Os06g02960 The application is characterized by: The gene LOC_Os06g02960 The exon nucleotides are shown in SEQ ID NO.

3.

4. The gene according to claim 1 LOC_Os06g02960 The application is characterized by: The gene LOC_Os06g02960 The encoded amino acid sequence is shown in SEQ ID NO.

4.

5. The gene according to any one of claims 1 to 4 LOC_Os06g02960 The application is characterized by: The gene editing vector used for the knockout is a CRISPR / Cas9 gene editing vector; the target sequence of the CRISPR / Cas9 gene editing vector is: 5'-AGCAGTACAAGTAGGAGCGG-3'.

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