Application of rice blast disease resistance related gene OsCBP606

Abstract

The invention discloses application of a rice blast disease resistance related gene OsCBP606, and belongs to the technical field of plant genetic engineering. The expression quantity of the OsCBP606 is increased after the OsCBP606 is induced by pathogenic bacteria of rice blast in susceptible and disease-resistant materials. After the gene is knocked out, the rice blast resistance can be improved. The OsCBP606 editing site sequence is connected to a gene editing vector pRGEB32S to transform rice, and the result shows that the gene can negatively regulate and control the rice blast resistance. A gene editing carrier constructed by the invention can be applied to rice blast-resistant breeding of rice. According to the invention, the action mechanism of the OsCBP606 can be better understood, the cloning of the OsCBP606 lays a foundation for further understanding of rice-rice blast pathogen interaction and a disease-resistant signal transduction pathway, and the OsCBP606 has a great application value in breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering and relates to the application of the rice blast disease resistance-related gene OsCBP606. Background Art

[0002] Rice (Oryza sativa L.) is one of the important food crops worldwide, and more than 50% of the population depends on rice as the main food. Maintaining the stable production and supply balance of rice is crucial for ensuring social stability and economic development. Rice blast, caused by Magnaporthe oryzae, is one of the three most destructive diseases of rice. Although traditional chemical agents can inhibit the occurrence of rice blast to a certain extent, their drug residues not only damage the ecological environment but also may threaten the health and safety of consumers. Long-term production practice has shown that exploring rice blast resistance genes and cultivating and promoting disease-resistant rice varieties is an economical, efficient, and eco-friendly prevention and control strategy.

[0003] To date, researchers have identified more than 100 rice blast resistance (R) genes and successfully cloned more than 30 genes. Previous identification of R genes mainly relied on map-based cloning technology, which was studied by constructing large segregating populations such as F2 populations, recombinant inbred lines (RILs), and doubled haploid populations. However, due to the limited genetic diversity between parents, this method can only analyze the variation of two alleles, and the process of constructing populations is time-consuming and laborious, which limits the research efficiency.

[0004] With the development of high-throughput sequencing technology, researchers can perform deep sequencing of a large number of genomes at a lower cost, which has greatly promoted the development of genome-wide association studies (GWAS). GWAS provides a powerful means for simultaneously exploring multiple genetic variations behind complex phenotypes. Based on the study of the rice integrated HapMap, in-depth analysis of rice blast resistance was carried out through GWAS, and multiple gene loci related to disease resistance were identified, such as bsr-d1, pb3, and pb4, etc., which provided important clues for the study of rice blast resistance.

[0005] Large-scale identification of the resistance of germplasm resources can provide more possibilities for the discovery of resistance genes or QTLs, and multiple resistance genes or QTLs can be pyramided through marker-assisted selection, which provides an important means for improving the broad-spectrum and durable resistance of rice. With the rapid development of sequencing technology and the significant reduction in cost, whole-genome resequencing combined with GWAS has been widely applied in the field of plant disease resistance research. However, due to the limitations of marker density and linkage disequilibrium (LD), it is difficult for GWAS to accurately locate target genes. Transcriptome analysis can effectively make up for this deficiency by detecting and comparing the expression levels of candidate genes with different genotypes, thereby further improving the identification efficiency of disease resistance genes.

[0006] The identification of disease resistance-related genes not only provides a basis for deeply revealing the disease resistance mechanism of rice and its interaction mechanism with pathogenic bacteria, but also lays an important foundation for the cultivation of disease-resistant varieties. Through in-depth research on these disease resistance genes, it not only helps to control and reduce the harm of rice blast to rice production, but also can significantly enhance the disease resistance of plants. These studies have important theoretical significance and application value in the fields of rice gene function analysis and disease-resistant breeding. Summary of the Invention

[0007] In order to overcome the disadvantages and deficiencies of the prior art, the purpose of the present invention is to provide the application of the rice blast resistance-related gene OsCBP606.

[0008] The present invention relates to the identification and cloning of plant resistance genes, and provides a new gene OsCBP606 that negatively regulates rice blast resistance. This gene is located on chromosome 12, and the gene locus number is LOC_Os12g36910. Its full-length genomic sequence is 4325bp (SEQ ID NO:1), including a 5′ untranslated region (5′UTR), 10 exons, 9 introns, and a 3′ untranslated region (3′UTR). Its cDNA full length is 1731bp (SEQ ID NO:2), encoding 576 amino acids. The protein sequence encoded by OsCBP606 is shown in SEQ ID NO:3.

[0009] This gene is divided into four different haplotypes in the natural population involved in the present invention. The deletion of this gene will improve rice blast resistance. After infection with Magnaporthe oryzae, the expression levels of this gene in both disease-resistant and susceptible germplasms increase. Different haplotypes of OsCBP606 in rice germplasm can be used or gene editing technologies such as CRISPR / Cas9 can be combined to perform site-directed editing of this gene to improve rice blast resistance.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] The application of the rice blast resistance-related gene OsCBP606 in the following (1) or (2):

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

[0013] (2) Application in cultivating transgenic rice with enhanced susceptibility or resistance to Magnaporthe oryzae;

[0014] Wherein, the protein sequence encoded by the rice blast resistance-related gene OsCBP606 is shown in SEQ ID NO:3.

[0015] Furthermore, the full-length genomic sequence of the rice blast resistance-related gene OsCBP606 is shown in SEQ ID NO: 1.

[0016] Furthermore, the full-length cDNA sequence of the rice blast resistance-related gene OsCBP606 is shown in SEQ ID NO: 2.

[0017] Furthermore, the application (1) is: by reducing or interfering with the expression of the rice blast resistance-related gene OsCBP606, or knocking out the rice blast resistance-related gene OsCBP606, the resistance of rice to Magnaporthe oryzae is enhanced.

[0018] Furthermore, the application (2) is: reducing or interfering with the expression of the rice blast resistance-related gene OsCBP606 in rice, or knocking out the rice blast resistance-related gene OsCBP606, to cultivate transgenic rice with enhanced resistance to Magnaporthe oryzae.

[0019] Furthermore, the reduction or interference is achieved by antisense RNA, siRNA or shRNA.

[0020] Furthermore, the knockout is achieved by a CRISPR / Cas9 gene editing vector.

[0021] Furthermore, the knockout is achieved by a host bacterium containing a CRISPR / Cas9 gene editing vector.

[0022] Furthermore, the target sequence of the CRISPR / Cas9 gene editing vector is: 5'-GCTCGAGTCCGCCATTAGCC-3'.

[0023] The application of primers for amplifying the above-mentioned rice blast resistance-related gene OsCBP606 in the identification and / or evaluation of rice blast resistance.

[0024] The present invention discovers that the expression level of the rice blast resistance-related gene OsCBP606 increases after being induced by Magnaporthe oryzae, and knocking out the disease-resistant genotype can further improve the blast resistance. A 20bp containing a PAM sequence is selected as the knockout target in the exon of OsCBP606, which is ligated with the U3-gRNA expression cassette and then constructed into the vector pRGEB32S-OsCBP606 with Cas9. After transforming Agrobacterium, the callus of rice is infected. The knockout plants are obtained by hygromycin screening. The results show that the deletion of this gene can significantly improve the blast resistance of rice. The gene editing vector pRGEB32S-OsCBP606 of the present invention can be used in rice blast resistance breeding.

[0025] The inventors of the present invention first identified 7 QTLs through a genome-wide association study (GWAS) of rice blast resistance in 236 rice germplasms. Further, by referring to the transcriptome sequencing data of the core genes involved in the immune response in rice, it was found that the expression level of OsCBP606 increased in both susceptible and resistant materials after infection with Magnaporthe oryzae. Through haplotype analysis, it was found that haplotype 1 was more susceptible than haplotype 2 and haplotype 3. Knocking out this gene in Zhonghua 11 significantly improved the resistance of rice to rice blast. It was predicted through the STRING database (https: / / cn.string-db.org / cgi / input?sessionId=bMlAiMzFQ4R6&input_page_show_search=on) that the protein encoded by this gene might interact with the transcriptional and export factor ENY2, the transcription initiation factor TFIID10, and the NLR protein. Through the Rice Expression Database (https: / / ricexpro.dna.affrc.go.jp / ), it was found that this gene was expressed in various parts of rice, and its expression level increased in rice leaves after inoculation with Magnaporthe oryzae.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] The method of the present invention identified a rice blast resistance gene OsCBP606 from rice by using genome-wide association analysis and transcriptome sequencing analysis. It was confirmed through the CRISPR / Cas9 gene editing technology that OsCBP606 is involved in the defense response of rice to Magnaporthe oryzae and is an important negative regulatory gene involved in rice disease resistance. The present invention helps to better understand the mechanism of action of OsCBP606. The identification of OsCBP606 lays a foundation for further understanding the interaction between rice and pathogens and has great application value in breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a diagram showing the investigation results of rice blast resistance of 236 rice germplasms;

[0029] Figure 2 It is a manhattan plot and a Q-Q plot of genome-wide association analysis; the brown * represents peak-SNP;

[0030] Figure 3 It is a diagram showing the change in the expression level of OsCBP606 in response to infection with Magnaporthe oryzae;

[0031] Figure 4 It is a diagram of haplotype analysis of OsCBP606;

[0032] Figure 5 It is a diagram showing the identification of the OsCBP606 deletion mutant and the sequencing results of the target site;

[0033] Figure 6 It is the phenotypic diagram of the OsCBP606 mutant; WT is Zhonghua 11 (ZH11). Specific implementation manners

[0034] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0035] All kinds of raw materials and various equipment used in the present invention are all conventional commercially available products and can be directly obtained through market purchase without special instructions.

[0036] The primer sequences used in the embodiments of the present invention were all synthesized by Shanghai Sangon Biotech Co., Ltd.

[0037] Lijiangxintuanheigu (LTH) described in the embodiments of the present invention has been disclosed in the literature "Research and Utilization of the Universal Susceptibility Characteristics of Rice Variety Lijiangxintuanheigu [J]. Scientia Agricultura Sinica, 2001, 01: 121-121."

[0038] The highly blast-resistant rice strain Pilk-H4 NIL described in the embodiments of the present invention has been disclosed in the literature "Cloning of the Rice Blast Resistance Protein Pik2-H4 Gene and Screening of Its Interaction Proteins [J]. Guangdong Agricultural Sciences, 2014, (04): 156-160."

[0039] The Magnaporthe oryzae GDYJ7 described in the embodiments of the present invention has been disclosed in the literature "Wu Pengzhi, Identification and Preliminary Functional Research of Rice MicroRNAs Responding to Magnaporthe oryzae Infection (D), 2019."

[0040] The Magnaporthe oryzae Zhong1 described in the embodiments of the present invention has been disclosed in the literature "Yu, S.; Li, S.; Wang, W.; Tang, D. OsCAMTA3 Negatively Regulates Disease Resistance to Magnaporthe oryzae by Associating with OsCAMTAPL in Rice. Int. J. Mol. Sci. 2024, 25, 5049."

[0041] Example 1: Resistance investigation and genome-wide association analysis of 236 rice germplasms

[0042] 1) Resistance investigation of 236 rice germplasms

[0043] For all germplasms, 20 pre-germinated rice seeds were sown in flat seedling trays filled with soil, and grown for about 14 days until the three-leaf and one-heart stage for inoculation with Magnaporthe oryzae (GDYJ7). The pathogenic fungus of rice blast was cultured in an inverted manner on CM medium in an incubator at 28 °C for 3 - 4 days for activation, then transferred to a new CM medium and cultured in an inverted manner for 7 days until it was nearly full of the dish. 3 - 5 mL of sterile water was added, and the mycelium was broken into small fragments with a spreading rod. The mycelial suspension was aspirated and transferred to a tomato-oat sporulation plate, spread evenly and air-dried, and cultured for 4 - 7 days. 3 mL of 0.05% Tween aqueous solution was added to each petri dish, and the spores were gently washed off with a scraper, and then the mycelium was filtered through two layers of gauze to collect the spore suspension. After mixing, the concentration of the spore suspension was adjusted to 5×10 5 conidia / mL using a hemocytometer. For each rice material, 6 leaves about 6 cm long were cut and placed in a petri dish containing 25 mL of 6-BA solution with a concentration of 10 μg / mL, and treated in the dark for 24 h. Then, after 5 days of normal light at 28 °C, the disease incidence was observed and recorded. Image-Pro 6.0 software was used to calculate the lesion length and area. Using the susceptible control Lijiangxintuanheigu (LTH) as a control, the ratio of the average value of the lesion length of different germplasms to the average value of the lesion length of LTH was calculated as the data for evaluating rice blast resistance. The disease grades were as follows: highly resistant (HR): no brown lesions; resistant (R): only needle-shaped brown lesions with a length less than 1 mm; moderately resistant (MR): lesion length 1.0 - 2.0 mm; moderately susceptible (MS): typical spindle-shaped lesions, and the lesion area accounted for 2.0 - 10.0% of the leaf area; susceptible (S): typical spindle-shaped lesions, and the lesion area accounted for 10.1 - 50.0% of the leaf area; highly susceptible (HS): typical spindle-shaped lesions, and the lesion area accounted for more than 50% of the leaf area.

[0044] The resistance evaluation data of all germplasms are as Figure 1 shown.

[0045] 2) Genome-wide association study

[0046] The whole-genome resequencing results of 236 rice germplasms were used as genotypes for genome-wide association analysis with rice blast resistance. The analysis software was Emmax (Kang et al., 2010). The individual kinship coefficient matrix (kinship matrix) was used as a covariate to correct the association results, and the association threshold was 7 (based on -log 10 (1 / 335897). Significantly associated SNP loci were screened according to the threshold, and a QTL interval with a range of 200 kb upstream and downstream of the significantly associated loci was used as a QTL interval. Each QTL contained at least two or more significant SNPs.

[0047] The results are as Figure 2As shown, a total of 7 QTLs were identified. Candidate genes were determined by searching the region 200 kb upstream of the most significant SNP with the smallest physical position to 200 kb downstream of the most significant SNP with the largest physical position in each QTL through core genes involved in immune responses in rice. The candidate gene OsCBP606 was mapped at qMZ12.1, and at the same time, the identified blast resistance genes OsPR10, OsPR10a, and PR10b were also mapped. The Q-Q plot indicated that this model could effectively control the effects of population structure and kinship on the results of the association analysis, and the significance of the associated loci was relatively strong.

[0048] 3) OsCBP606 responds to Magnaporthe oryzae infection

[0049] To further verify the role of OsCBP606 in blast resistance, qRT-PCR technology was used to analyze the expression changes of OsCBP606 in the resistant rice variety Pik-H4 NIL and the susceptible rice variety LTH. The primers used were as follows:

[0050] qRT-OsCBP606-F: 5’-GCCATTAGCCGGATTCCAGA-3’

[0051] qRT-OsCBP606-R: 5’-AGCTTGTACCTTGGAGGCTG-3’.

[0052] The results were as Figure 3 shown. The expression patterns of OsCBP606 in response to Magnaporthe oryzae infection were significantly different between the two varieties. In the susceptible variety LTH, the level of OsCBP606 decreased significantly at 6 h after inoculation and remained at a low level until 24 h, then increased and reached a peak at 36 h. In contrast, in the resistant variety Pik-H4 NIL, the accumulation of OsCBP606 increased sharply at 12 h after inoculation and reached a peak at 36 h after inoculation. This finding further confirmed that OsCBP606 responds to Magnaporthe oryzae infection, indicating that OsCBP606 is involved in the immune-related processes of rice.

[0053] Example 2: Haplotype analysis of OsCBP606

[0054] The variant site information of the genomic region of the target gene of all materials and the promoter region of 2000 bp upstream was obtained using the OE Biotech bioinformatics cloud platform, and all SNP sites were classified in the manner described in "Genome-wide association study using whole-genome sequencing rapidly identifies new genes influencing agronomic traits in rice. Nature Genetics, 2016, 48(8):927-934". Haplotype analysis was performed on the significant SNPs that could cause non-synonymous mutations. Haplotypes with a sample size of less than 5 were removed, the phenotypic data of the rice germplasms corresponding to the haplotypes were sorted out, box plots of the haplotypes were drawn using GraphPad, and variance analysis was performed.

[0055] The results are as Figure 4 shown. The key SNPs of OsCBP606 are mainly concentrated in the exon region, which can be divided into 4 haplotypes, and the average resistance of haplotype 1 is lower than that of haplotype 2, haplotype 3, and haplotype 4.

[0056] Example 3: Construction of the OsCBP606 gene editing vector and phenotypic identification of mutants

[0057] The OsCBP606 knockout vector was constructed by modifying 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") to pRGEB32S and then further constructing it. That is, the connection sequence between the U3 promoter and gRNA in pRGEB32 was changed to a double digestion site sequence. Using the CDS sequence of OsCBP606, a suitable target site (target sequence: 5'-GCTCGAG TCCGCCATTAGCC-3') was searched on the CRISPR / Cas9 online target site design website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). Primers OsCBP606-ko-F and OsCBP606-ko-R were designed according to the target site, and touchdown PCR was performed with the synthesized primers to complementarily form a target fragment with pRGEB32S homologous arms. The Cas9 protein expression vector pRGEB32S was double digested with BamHⅠ and KpnⅠ, and the obtained fragment with the target site was recombinantly ligated with the vector. After transforming the recombinant vector into Escherichia coli DH5α, sequencing identification was carried out. 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 sites of the positive plants were detected by Sanger sequencing, and were denoted as OsCBP606-ko1 and OsCBP606-ko2 according to the mutation types ( Figure 5 ). The primer sequences used are as follows:

[0058] OsCBP606-ko-F: 5'-TGCAGATGATCCGTGGCAGCTCGAGTCCGCCATTAGCCGTTTTAGAGCTAGAAATAG-3'

[0059] OsCBP606-ko-R: 5'-CTATTTCTAGCTCTAAAACGGCTAATGGCGGACTCGAGCTGCCACGGATCATCTGCA-3'.

[0060] The correctly identified OsCBP606-ko mutants were inoculated with Magnaporthe oryzae (Zhong1) at the three-leaf and one-heart stage, and the leaf lesion length and lesion area were observed 5 days after inoculation.

[0061] The results are as Figure 6As shown, compared with wild-type plants, the lesion length and lesion area of OsCBP606-ko are relatively small. This result indicates that OsCBP606 negatively regulates rice blast resistance. Knocking out OsCBP606 can significantly improve rice blast resistance and thus significantly reduce the occurrence of rice blast. The gene editing vector constructed by the present invention can be applied to rice blast resistance breeding.

[0062] 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of the rice blast resistance-related gene OsCBP606 in the following (1) or (2): (1) Application in regulating rice blast resistance; (2) Application in breeding transgenic rice with enhanced susceptibility or resistance to rice blast fungus; in, The protein sequence encoded by the rice blast resistance-related gene OsCBP606 is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that: The full-length genome sequence of the rice blast resistance-related gene OsCBP606 is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that: The full-length cDNA sequence of the rice blast resistance-related gene OsCBP606 is shown in SEQ ID NO:

2.

4. The use according to claim 1, characterized in that: The application (1) is to enhance the resistance of rice to rice blast fungus by reducing or interfering with the expression of the rice blast resistance-related gene OsCBP606, or knocking out the rice blast resistance-related gene OsCBP606.

5. The use according to claim 1, characterized in that: The application (2) is to reduce or interfere with the expression of the rice blast resistance-related gene OsCBP606 in rice, or to knock out the rice blast resistance-related gene OsCBP606, so as to cultivate transgenic rice with enhanced resistance to rice blast fungus.

6. The use according to claim 4 or 5, characterized in that: The reduction or interference is achieved by antisense RNA, siRNA or shRNA.

7. The use according to claim 4 or 5, characterized in that: The knockout is achieved through CRISPR / Cas9 gene editing vector.

8. The use according to claim 7, characterized in that: The knockout is achieved by a host bacterium containing a CRISPR / Cas9 gene editing vector.

9. The use according to claim 7, characterized in that: The target sequence of the CRISPR / Cas9 gene editing vector is: 5'-GCTCGAGTCCGCCATTAGCC-3'.

10. Use of primers for amplifying the rice blast resistance-related gene OsCBP606 according to any one of claims 1 to 3 in identifying and / or evaluating rice blast resistance.

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