Application of RBL3 gene in regulating rice immunity level and / or rice disease resistance

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to the application of RBL3 gene in regulating rice immunity level and / or rice disease resistance. The present invention identifies a broad-spectrum disease resistance gene RBL3, which belongs to a member of the CDS gene family and encodes a CDP-DAG synthase, which regulates rice growth and immunity level by regulating rice lipid metabolism activity. By gene editing technology, the RBL3 gene in rice is knocked out, and it is found that rice shows obvious broad-spectrum disease resistance to rice blast and bacterial blight. In addition, the gene is highly conserved in crops, and can be widely promoted in multiple species by means of gene editing. Compared with traditional overexpression of disease resistance genes, it can reduce breeding time, improve crop disease resistance level, yield and quality, break species boundaries, and have stronger universality. It has huge disease resistance breeding application potential and can be widely used in disease resistance research of various crops.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of RBL3 gene in regulating rice immunity level and / or rice disease resistance. Background Art

[0002] Rice blast is one of the most serious fungal diseases that harm rice production. It is a common disease in rice fields, with the characteristics of long-term damage, multiple infected parts and diverse symptoms. Rice blast can occur throughout the entire growth period of rice, mainly harming seedlings, leaves, ears, nodes and other parts. Depending on the affected parts, it can cause seedling blast, leaf blast, node blast, ear neck blast and grain blast.

[0003] At present, the prevention and control of rice blast disease mainly relies on planting disease-resistant varieties and using chemical pesticides. With the improvement of rice genome sequencing, the research on rice resistance genes has also increased. Since the 1960s, researchers have screened the rice blast resistance genes Pia, Pii and Pik from breeding materials, and now have cultivated new rice varieties with excellent resistance, such as Fyou 498, Yixiangyou 2115, Zhongke 804, and Longliangyou 3189. However, with global climate change, large-scale agricultural production and increased international grain trade, the evolution and spread of pathogen populations are accelerating. Single disease-resistant varieties gradually lose their resistance after many years of planting, and the introduction of multiple R genes (Resistance genes) is likely to affect grain yield. Therefore, it is crucial to clone durable disease resistance genes and cultivate high-quality materials with broad-spectrum disease resistance and no yield loss. However, R genes are now facing the current situation of unclear mechanism analysis and scarce resources. At the same time, the resistance of R genes expires in about 4 years. In addition, fungi are also constantly evolving and mutating rapidly during their growth. Even when multiple R genes are superimposed together, it still takes longer and costs more to cultivate high-quality rice. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of RBL3 gene in regulating rice immunity level and / or rice disease resistance. The present invention identifies the RBL3 gene and obtains rbl3 mutants based on the CRISPR / Cas9 system. The mutants have broad-spectrum disease resistance and can help researchers to screen high-quality disease-resistant materials more quickly.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of the RBL3 gene in one or more of 1) to 4), wherein the amino acid sequence encoded by the RBL3 gene is shown in SEQ ID NO.2:

[0007] 1) Regulate rice lipid metabolism; 2) Regulate rice growth; 3) Regulate rice immunity level; 4) Regulate rice disease resistance.

[0008] Preferably, the regulating rice disease resistance comprises knocking out the RBL3 gene to improve rice disease resistance.

[0009] Preferably, the diseases targeted by the rice disease resistance include rice blast and / or bacterial blight.

[0010] Preferably, the regulation of rice lipid metabolism activity includes: knocking out the RBL3 gene to increase the phosphatidic acid content of rice, knocking out the RBL3 gene to reduce the phosphatidylinositol content of rice, knocking out the RBL3 gene to reduce the phosphatidylserine content of rice, and knocking out the RBL3 gene to reduce the phosphatidylinositol phosphate content of rice. One or more.

[0011] Preferably, the regulating the immunity level of rice includes knocking out the RBL3 gene to improve the immunity level of rice.

[0012] The present invention provides a recombinant vector for knocking out an RBL3 gene, comprising a target sequence and a CRISPR / Cas9 vector; the nucleotide sequence of the target sequence is shown in SEQ ID NO.4; and the amino acid sequence encoded by the RBL3 gene is shown in SEQ ID NO.2.

[0013] Preferably, the CRISPR / Cas9 vector comprises a pRGEB32 vector.

[0014] Preferably, the recombinant vector for knocking out the RBL3 gene comprises the sequence shown in SEQ ID NO.23 and a double-enzyme-digested pRGEB32 vector; the enzymes used for the double-enzyme-digestion are FOKI and BSAI.

[0015] The present invention provides the use of the recombinant vector described in the above technical solution in improving the immunity level of rice and / or improving the disease resistance of rice.

[0016] Preferably, the diseases targeted by the rice disease resistance include rice blast and / or bacterial blight.

[0017] Beneficial effects:

[0018] The present invention identifies a broad-spectrum disease resistance gene RBL3, which belongs to a member of the CDS gene family and encodes a CDP-DAG synthase, which regulates rice growth and immunity by regulating rice lipid metabolism activities. Through gene editing technology, the RBL3 gene in rice is knocked out, and it is found that rice shows obvious broad-spectrum disease resistance to rice blast and white leaf blight. In addition, this gene is highly conserved in crops, and can be widely promoted in multiple species through gene editing. Compared with traditional over-expressed disease resistance genes, it can reduce breeding time, improve crop disease resistance level, yield and quality, break species boundaries, and have stronger universality. It has huge disease resistance breeding application potential and can be widely used in disease resistance research of various crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 This is the phylogenetic tree of RBL3 homologous proteins in different species;

[0021] Figure 2 Validation of RBL3 functional results for yeast cds1 complementation;

[0022] Figure 3 This is a schematic diagram of the recombinant CRISPR vector of Example 3;

[0023] Figure 4 Schematic diagram of gene editing and phenotypic comparison of rbl3 mutant and wild type (WT); wherein a is a schematic diagram of gene editing and b is a phenotypic comparison;

[0024] Figure 5 The results of the basal reactive oxygen species measurement in the rbl3 mutant and wild type;

[0025] Figure 6 The results of the disease occurrence of Xanthomonas oryzae and Magnaporthe oryzae in the rbl3 mutant and wild type;

[0026] Figure 7 The results of the expression level determination of disease resistance-related genes in rbl3 mutant and wild type;

[0027] Figure 8 The experimental results for regulating lipid metabolism activities in rice;

[0028] Fig. 9 The growth phenotype of the rbl1 mutant and the results of its pathogenicity to Xanthomonas oryzae and Magnaporthe oryzae are shown;

[0029] Fig.10 The result is that overexpression of RBL3 in the rbl1 mutant can complement the lesion-like phenotype;

[0030] Fig.11 Statistical results of plant height, tillering and panicle length of wild type and rbl3 mutant. DETAILED DESCRIPTION

[0031] The present invention provides the use of the RBL3 gene in one or more of 1) to 4), wherein the amino acid sequence encoded by the RBL3 gene is shown in SEQ ID NO.2:

[0032] 1) Regulate rice lipid metabolism; 2) Regulate rice growth; 3) Regulate rice immunity level; 4) Regulate rice disease resistance.

[0033] In the present invention, the regulation of rice lipid metabolism activity preferably includes: knocking out the RBL3 gene to increase the phosphatidic acid content of rice, knocking out the RBL3 gene to reduce the phosphatidylinositol content of rice, knocking out the RBL3 gene to reduce the phosphatidylserine content of rice, and knocking out the RBL3 gene to reduce the phosphatidylinositol phosphate content of rice. One or more of the following; the regulation of rice growth preferably includes: expressing the RBL3 gene can complement the growth defect of the rice disease spot mutant, that is, there is redundancy between the RBL3 protein and RBL1 functions in rice; the regulation of rice immunity level preferably includes knocking out the RBL3 gene to increase the rice immunity level; the regulation of rice disease resistance preferably includes knocking out the RBL3 gene to increase the rice disease resistance; the diseases targeted by the rice disease resistance preferably include rice blast and / or bacterial blight; the coding sequence of the RBL3 gene is preferably as shown in SEQ ID NO.1, and the sequence information is as follows:

[0034]

[0035] The RBL3 protein sequence is shown in SEQ ID NO. 2, specifically as follows: MPHVRSAAERDNGSGGDVTPGTPSPTHGARVRQRKRSSDAPSDVNKTNGANLLLNDQNKYKSMLIRTYSSLWMMAGFVFLIYMGHLYIWAMVVVIQIFMASELFNLLRKANEDRQLPGFRLLNWHFFFTAMLFAYGRFLSRQLVNTVTSDKLLYKLVSGLIKYQMFICYFLYIAGFVWFILTLKKKAYKYQFSQYAWTHMILLMVFAQS SFTVANIYEGMFWFLLPASLIAINDVAAYFFGFFFGKTPLIKLSPKKTWEGFLGASVTTMLSAFVLANFMGHFQWLTCPRKDLSTGWLHCDPGSIFTPESYDLPGWIPWRE VAIMPIQWHALALGLFASIIAPFGGFFASGFKRAFKFKDFGDSIPGHGGFTDRMDCQMVMAVFAYIYYQSFVMVQDLSVETIMEQILRNLTFEEQHDLYEQLGKLLTRGN.

[0036] The present invention identifies a broad-spectrum disease resistance gene RBL3, which belongs to a member of the CDS gene family and encodes a CDP-DAG synthase, which regulates rice growth and immunity by regulating rice lipid metabolism activities. Through gene editing technology, the RBL3 gene in rice is knocked out, and it is found that rice shows obvious broad-spectrum disease resistance to rice blast and white leaf blight. In addition, this gene is highly conserved in crops, and can be widely promoted in multiple species through gene editing. Compared with traditional over-expressed disease resistance genes, it can reduce breeding time, improve crop disease resistance level, yield and quality, break species boundaries, and have stronger universality. It has huge disease resistance breeding application potential and can be widely used in disease resistance research of various crops.

[0037] The present invention provides a recombinant vector for knocking out an RBL3 gene, comprising a target sequence and a CRISPR / Cas9 vector; the nucleotide sequence of the target sequence is shown in SEQ ID NO.4; and the amino acid sequence encoded by the RBL3 gene is shown in SEQ ID NO.2.

[0038] In the present invention, the CRISPR / Cas9 vector preferably includes a pRGEB32 vector; the recombinant vector for knocking out the RBL3 gene preferably includes a sequence shown in SEQ ID NO.23 and a double-enzyme-digested pRGEB32 vector; the enzymes used for the double-enzyme-digestion are FOKI and BSAI.

[0039] The present invention provides the use of the recombinant vector described in the above technical solution in improving the immunity level of rice and / or improving the disease resistance of rice. In the present invention, the diseases targeted by the rice disease resistance preferably include rice blast and / or bacterial blight.

[0040] To further illustrate the present invention, the application of the RBL3 gene provided by the present invention in regulating rice immunity level and / or rice disease resistance is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] Conservation analysis of RBL3 protein

[0043] In order to explore the biological function of the RBL3 gene (the coding region sequence is shown in SEQ ID NO.1), the present invention first analyzed the sequence specificity of the amino acid sequence (SEQ ID NO.2) encoded by the RBL3 gene in different biological species. The present invention retrieved all RBL3 homologous proteins in plants, animals, and microorganisms through the Protein BLAST function on NCBI, and compared the RBL3 amino acid sequences in different species using sequence analysis tools such as MEGA11. The results are shown in Table 1. Figure 1 The results showed that RBL3 has a conserved CTP-transf_1 domain, in which the amino acid sequence of the C-terminal functional domain of the RBL protein (KDXXXXXPGHGGXXDRXD, SEQ ID NO.3, X is any amino acid) is highly conserved in all animals, plants and microorganisms. Through gene editing, it can be widely promoted in multiple species to accelerate disease-resistant breeding research.

[0044] Example 2

[0045] In order to further verify whether the RBL3 protein in rice has the function of CDP-DAG synthase (RBL3), the present invention carried out functional complementation verification in Saccharomyces cerevisiae. Since Cds1 mutation in yeast is lethal, the present invention refers to the method described in the reference [Sha G, Sun P, Kong X, et al. Genome editing of a rice CDP-DAG synthase confers multipathogen resistance [J]. Nature, 2023, 618 (7967): 1017-1023.], firstly constructed a galactose-inducible RBL3 expression vector, transformed the yeast BY4741 strain, and screened the cds1-CoRBL3 positive strain on YPGal medium, named Sccds1 / OsCDS3. The Sccds1 / OsCDS3 strain and the yeast wild-type strain BY4741 (WT) were cultured on YPGal medium and YPD medium, respectively, and the culture results are shown in Figure 2 .

[0046] The galactose-inducible RBL3 expression vector was constructed as follows:

[0047] The primer pairing was PYES2 / RBL3-F and PYES2 / RBL3-R, and PCR amplification was performed using the cDNA sequence of Kitaake rice as a template; the sequence information is as follows:

[0048] PYES2 / RBL3-F: 5'-ACGATGACGATAAGGTACCTAATGCCCCATGTTAGATCCG-3', SEQ IDNO.24;

[0049] PYES2 / RBL3-R: 5'-ATATCTGCAGAATTCCACCACACTTCAGTTCCCTCTCGTCAG-3', SEQ ID NO. 25.

[0050] The reaction conditions of the PCR amplification were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2 min, 32 cycles;

[0051] The reaction system of the PCR amplification is: 5.2 μL of ddH2O, 10 μL of 2×Phanta Mix, 0.4 μL of dNTP, 1 μL of primer F, 1 μL of primer R, 2 μL of template, and 0.4 μL of Phanta enzyme.

[0052] The amplified cDNA fragment was ligated with the pYES2 vector digested with BamHI (enzyme cleavage site: GGATCC) to obtain the RBL3 expression vector.

[0053] Depend on Figure 2 It can be seen that on the YPD medium, the yeast cdsC strain could not grow normally because the transferred rice RBL3 gene was not expressed, while on the YPGal medium, the added galactose induced the expression of rice RBL3 and the yeast could grow normally. It can be seen that expressing the rice RBL3 gene in the yeast cds1 mutant can complement the growth defect of the yeast cds1 mutant, that is, the RBL3 protein in rice has the function of CDP-DAG synthase.

[0054] Example 3

[0055] The CRISPR vector construction method is derived from the article [Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system [J]. National Academy of Sciences, 2015 (11). DOI: 10.1073 / PNAS.1420294112.]. As follows, the sequences of the primers in the following steps are the same as those in the public article unless otherwise specified:

[0056] (1) Primer design:

[0057] First, enter the RBL3 gene LOC number: LOC_Os10g17990 in the rice gene database website (http: / / www.ricedata.cn / gene / ). Find a suitable target site based on the RBL3 gene:

[0058] The editing target sequence was designed as gRNA-30: 5'-GCCATTGTCCCTTTCAGCGG-3' (SEQ ID NO.4). The target is located in the second exon of the gene and has a low off-target rate. The gRNA scaffold sequence was connected to the target: 5'-GTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3', SEQ ID NO.5. The primers designed based on the target are as follows:

[0059] RBL3-F: 5'-TAGGTCTCCCCCTTTCAGCGGgttttagagctagaa-3', SEQ ID NO.6;

[0060] RBL3-R: 5'-CGGGTCTCAAGGGACAATGGCtgcaccagccggg-3', SEQ ID NO.7; wherein, 5'-gttttag agctagaa-3' (SEQ ID NO.21) and 5'-tgcaccagccggg-3' (SEQ ID NO.22) are gRNA scaffold sequences.

[0061] (2) One round of PCR: The primer pairs were L5AD5-F and RBL3-R, and RBL3-F and L3AD3-R, and PCR amplification was performed using the artificially synthesized PGTR sequence as a template; the sequence information is as follows:

[0062] L5AD5-F: 5'-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCAACAAAGCACCAGTGG-3', SEQ IDNO.8;

[0063] L3AD3-R: 5'-TAGGTCTCCAAACGGATGAGCGACAGCAAACAAAAAAAAAAGCACCGACTC G-3', SEQ ID NO.9;

[0064] PGTR sequence: 5'-CCTCTATATCTGGGCCATGGTGGGTTTTAGAGCTAGAAATAGCAAGTTAAAAT AAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3', SEQ ID NO. 10.

[0065] The reaction conditions of the PCR amplification are: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 32 cycles; extension at 72°C for 5 min;

[0066] The reaction system of the PCR amplification is: 6.2 μL of ddH2O, 10 μL of 2×Phanta Mix, 0.4 μL of dNTP, 1 μL of primer F, 1 μL of primer R, 1 μL of template, and 0.4 μL of Phanta enzyme.

[0067] (3) GG reaction: The PCR products obtained by amplification were purified and then subjected to GG reaction; the reaction system of the GG reaction was: 7 μL (25-50 ng, mass ratio of 1:1) of two PCR products, 10 μL of 2×T4 ligase buffer (NEB), 2 μL of BSA, 0.5 μL of BsaI (10 U / μL, NEB), and 0.5 μL of T4 DNA ligase; the reaction conditions of the GG reaction were: 37°C for 5 min, 20°C for 10 min, 40 cycles; 20°C for 1 h.

[0068] (4) GG PCR: The reaction product was diluted with 180 μL of double distilled water and then the target fragment was amplified using S5AD5-F and S3AD3-R, and the PCR product was purified; the primer sequences are as follows:

[0069] S5AD5-F: 5'-CGGGTCTCAGGCAGGATGGGGCAGTCTGGGCA-3', SEQ ID NO. 11;

[0070] S3AD3-R: 5'-TAGGTCTCCAAACGGATGAGCGACAGAAAC-3', SEQ ID NO. 12;

[0071] The reaction system was the same as step (2); the reaction procedure was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min.

[0072] (5) Enzyme digestion and ligation: The fragment amplified in step (4) and the pRGEB32 vector were digested with FOKI and BSAI, respectively, and reacted at 37°C for 2 hours. After the digestion, the digested fragment containing the target sequence of the RBL3 gene (the sequence after digestion is shown in SEQ ID NO.23) and the digested pRGEB32 vector were ligated and then transformed into E. coli Top10. The recombinant CRISPR vector was obtained by PCR screening of positive clones (amplification primer pRGEB32 / F: 5'-aaaagcatttcgtagtgggc-3', SEQ ID NO.26; pRGEB32 / R: 5'-ttctggctggtttgttggtc-3', SEQ ID NO.27); the sequence shown in SEQ ID NO.23 is as follows: 5'-GGGTCTCAGGCAGGATGGGCAGTCTGGGCAaacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacaga cccgggttcgattcccggctggtgcaGCCATTGTCCCTTTCAGCGGgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTTTTTGTTTGCTGTCGCTCATCCGTTTGGAGACCTA-3'.

[0073] (6) Transformation: The recombinant CRISPR vector was commissioned to Wuhan Boyuan Biotechnology Co., Ltd. to transform rice Kitaake materials through Agrobacterium-mediated genetic transformation technology, and the genotype of the T0 generation gene-edited rice materials was identified by Sanger sequencing. The homozygous mutant material of rbl3 was obtained by screening in the T1 generation ( Figure 4 ).

[0074] Example 4

[0075] Fungal cell walls are rich in chitin. Researchers usually treat plant tissues with chitin to simulate the immune response (PTI) triggered by pathogen-associated molecular patterns, and preliminarily judge the level of plant disease resistance by the degree of active oxygen burst. The present invention treats wild-type and mutant seedling leaves with chitin, and observes the active oxygen burst by chemiluminescence. The specific method is as follows:

[0076] The seeds of wild-type rice Kitaake and mutant rice rbl3 were cultured on 1 / 2MS sterile medium. After 12 days, the seedling leaves were cut into small pieces (3mm×3mm), and the cut leaves were placed in a 96-well plate. 100μL of sterile double-distilled water was added to each well for 8-10 hours of recovery. The recovery solution was aspirated and 100μL of reaction solution was added. The reaction solution contained 50μM / L luminol, 10μg / mL peroxidase (HRP) and 8nM chitin. The sample added with the reaction solution was placed in a SPARK-10M microplate reader (TECAN), and the measurement was started immediately. The chemiluminescence was measured at 500ms intervals within 40 minutes. Three biological replicates were set for each sample, and each replicate contained 8 technical replicates. Sterile double-distilled water treatment was used as a negative control (mock). The results are shown in Figure 5 .

[0077] Depend on Figure 5 It can be seen that the basal reactive oxygen content of the rbl3 mutant was significantly higher than that of the wild type, and after chitin treatment, the reactive oxygen in the mutant leaves accumulated rapidly.

[0078] Example 5

[0079] Since the mutant can easily trigger apoptosis by itself, it is difficult for pathogens to expand their infection in the body, and they usually show high disease resistance. The present invention identified the disease resistance of the rbl3 mutant inoculated with bacterial blight and rice blast. The steps are as follows:

[0080] Identification of Rice Blast Resistance

[0081] (1) Cultivation of rice blast fungus

[0082] The paper pieces containing rice blast fungus (ZB25) spores were inoculated on oat culture medium and cultured in the dark at about 25°C for 2-3 days. After the mycelium germinated and grew out, light culture was carried out. Within two weeks, when the rice blast fungus filled the entire culture dish, the mycelium (containing a large number of conidia) was scraped and washed with distilled water containing 0.01% Tween-20, and filtered with double gauze. The spore concentration was calculated using a microscope and a blood cell counting plate, and the concentration of the spore suspension was adjusted to 2×10 5 spores / mL, used for rice blast inoculation; the rice blast fungus (ZB25) is derived from Huazhong Agricultural University and is disclosed in the literature [Sha G, Sun P, Kong X, et al. Genome editing of a rice CDP-DAG synthase confers multipathogen resistance[J]. Nature, 2023, 618(7967): 1017-1023.].

[0083] (2) Vaccination method

[0084] When rice (rbl3 mutant or wild type) reached the four-leaf stage, a potential circular wound was artificially made on the second leaf of the seedling with a mouse ear punch, and 10 μL of bacterial droplets were pipetted onto the wound and sealed with transparent tape; 5 plants were inoculated per strain, and the inoculation was repeated 3 times independently; then the plants were transferred to 28°C high humidity conditions for continued growth, and the disease (phenotype and lesion area) was investigated 14 days after inoculation. The results are shown in Figure 6 In the figure, the scale is 1 cm.

[0085] Identification of resistance to bacterial blight

[0086] 1) Cultivation of bacterial blight pathogen

[0087] The bacterial blight pathogen (PXO99) was cultured in NB medium at 28°C for 2-3 days. When the bacterial blight pathogen grew all over the culture dish, the bacteria were scraped and mixed in sterilized ddH2O, and the OD was adjusted to 0.5 for inoculation. The bacterial blight pathogen (PXO99) was derived from Huazhong Agricultural University and was disclosed in the literature [Chu C, Huang R, Liu L, Tang G, Xiao J, Yoo H, Yuan M. The rice heavy-metal transporter OsNRAMP1 regulates disease resistance by modulating ROS homoeostasis. Plant Cell Environ. 2022Apr;45(4):1109-1126.doi:10.1111 / pce.14263.Epub 2022Feb 3.PMID:35040151.].

[0088] 2) Inoculation method

[0089] The prepared bacterial solution was applied to rice leaves (rbl3 mutant or wild type) at the 4-leaf stage (seedling stage) and the 7-leaf stage using scissors. Five plants were inoculated per strain, and the results were repeated 3 times. The length of the lesions and the length of the diseased leaves were investigated 14 days after inoculation to investigate the incidence of the disease. Figure 6 In middle b, the scale is 1 cm.

[0090] Depend on Figure 6 It can be seen that 14 days after inoculation, the bacterial blight fungus only expanded by 7 cm on the mutant leaves, which was significantly lower than the 14 cm of the wild type; the lesion area of ​​the rice blast fungus was significantly lower than that of the wild type.

[0091] Example 6

[0092] Identification of disease resistance gene expression levels in rbl3 mutant

[0093] In the long-term co-evolution process with pathogens, plants have established a variety of immune regulation methods to control the expression of disease resistance genes such as PR. The present invention takes RNA from 4-week-old rice (rbl3 mutant or wild type) leaves for analysis, and uses Actin as an internal reference gene for qRT-PCR analysis. The primer sequences are as follows:

[0094] qRT-ActinF: 5'-CAGGCCGTCCTCTCTCTGTA-3', SEQ ID NO.13;

[0095] qRT-ActinR: 5'-AAGGATAGCATGGGGGAGAG-3', SEQ ID NO. 14;

[0096] qRT-PR1aF: 5'-CGTGTCGGCGTGGGGTGT-3', SEQ ID NO.15;

[0097] qRT-PR1aR: 5'-GGCGAGTAGTTGCAGGTGATG-3', SEQ ID NO.16;

[0098] qRT-PR1bF: 5'-TACGCCAGCCAGAGGAGC-3', SEQ ID NO.17;

[0099] qRT-PR1bR: 5'-GCCGAACCCCAGAAGAGG-3', SEQ ID NO.18;

[0100] qRT-PR10F: 5'-GTCCGGGCACCATCTACACC-3', SEQ ID NO.19;

[0101] qRT-PR10R: 5'-CAAGCTTCGTCTCCGTCGAGT-3', SEQ ID NO. 20;

[0102] The reaction system of the qRT-PCR was: 2×ChamQ SYBR qPCR Master Mix 10 μL, F-primer (10 μM) 0.4 μL, R-primer (10 μM) 0.4 μL, cDNA 2 μL, ddH2O 7.2 μL.

[0103] The reaction procedure of the qRT-PCR was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, 40 cycles;

[0104] The results showed that the expression of immune-related genes in the leaves of the rbl3 mutant was significantly upregulated even when not infected by pathogens, which was several times higher than that of the wild type ( Figure 8 ). By analyzing the phenotype of the rbl3 mutant and factors related to rice disease resistance, it was verified that the mutant gene plays an important role in rice immunity and growth and development.

[0105] Example 7

[0106] 1. Regulating rice lipid metabolism

[0107] In order to analyze the phospholipid metabolism process regulated by the RBL3 protein, the present invention analyzed the changes in phospholipid metabolism in the rbl3 mutant. The experimental process is as follows:

[0108] Cut 5-week-old rice leaves into 3 cm small pieces, soak in 3 mL isopropanol (containing 0.01% BHT), place in a 75°C water bath for 15 minutes; add 3 mL chloroform / methanol (2:1) solution (containing 0.01% BHT), shake for 1 hour, collect the extract, repeat adding new chloroform-methanol mixture to continue extraction until all leaves turn white. Add 2 mL KCl (1M) solution to the extraction solution, vortex mix, centrifuge at 4°C, 12,000×g, and remove the aqueous phase.

[0109] The lower organic phase was dried with nitrogen and redissolved in chloroform (10 mg / mL). The lipidomics analysis method was carried out according to the steps in the published literature [Sha G, Sun P, Kong X, et al. Genome editing of arice CDP-DAG synthase confers multipathogen resistance [J]. Nature, 2023, 618 (7967): 1017-1023.], using a TripleTOF mass spectrometer (TripleTOF-MS / MS) in positive ion mode to collect ionization data and perform relative quantitative analysis by internal standard method (Avanti).

[0110] Results Figure 8 The results showed that the content of phosphatidic acid (PA) in the rbl3 mutant increased by 2 times compared with the wild type, while the contents of phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylinositol phosphate (PIP) also decreased significantly. These results indicate that the RBL3 protein is involved in the phospholipid metabolism process.

[0111] 2. Regulate rice growth:

[0112] The present invention screened a disease spot mutant rbl1 from the rice Kitaake fast neutron mutant library, which has enhanced resistance to the fungal disease rice blast and the bacterial disease bacterial blight. However, the growth is seriously affected, and the growth is extremely short, such as Fig. 9 As shown, Fig. 9 Derived from the literature [Sha G, Sun P, Kong X, et al. Genome editing of a rice CDP-DAG synthase confers multipathogen resistance [J]. Nature, 2023, 618 (7967): 1017-1023.], wherein a, growth phenotypes of wild type KitaakeX (WT) and rbl1 mutant 40 days after sowing in the greenhouse, scale, 10 cm; b, leaf phenotypes of wild type and mutant in the adult stage, scale, 1 cm; c, phenotypes of wild type and mutant flag leaves 14 days after inoculation with bacterial blight; d, statistics of lesion length of each strain 14 days after inoculation with bacterial blight; e, phenotypes of wild type and mutant leaves 14 days after puncture inoculation with rice blast; f, statistics of lesion area (left) and fungal biomass (right) 14 days after inoculation with rice blast; the experimental steps for identifying resistance to rice blast and bacterial blight are detailed in Example 5.

[0113] The present invention refers to the method described in the reference document [Sha G, Sun P, Kong X, et al. Genome editing of a rice CDP-DAG synthase confers multipathogen resistance [J]. Nature, 2023, 618 (7967): 1017-1023.], firstly constructing an RBL3 overexpression vector, entrusting Wuhan Boyuan Biological Company to transform the rice mutant rbl1 material through Agrobacterium-mediated genetic transformation technology, and naming it OERBL3-rr. The results of OERBL3-rr are shown in Fig.10 .

[0114] The full-length genomic sequence of the RBL3 gene was cloned into the stable expression pRGV vector containing the maize Ubiquitin10 promoter. The RBL3 overexpression vector was constructed as follows:

[0115] The primer pairing was OE-RBL3 / F and OE-RBL3 / F, and PCR amplification was performed using the DNA sequence of Kitaake rice as a template; the sequence information is as follows:

[0116] OE-RBL3 / F: 5'-gatatccagatccagtgGGATCCATGCCCCATGTTAGATCCG-3', SEQ IDNO.28;

[0117] OE-RBL3 / R: 5'-gccgcactagtaagcttGGTACCTCAGTTCCCTCTCGTCAG-3', SEQ ID NO. 29.

[0118] The reaction conditions of the PCR amplification were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2 min, 32 cycles;

[0119] The reaction system of the PCR amplification is: 5.2 μL of ddH2O, 10 μL of 2×Phanta Mix, 0.4 μL of dNTP, 1 μL of primer F, 1 μL of primer R, 2 μL of template, and 0.4 μL of Phanta enzyme.

[0120] The amplified DNA fragment was ligated with the pRGV vector digested with Kpn1Ⅰ (enzyme cutting site: GGTACC) to obtain the RBL3 overexpression vector.

[0121] Depend on Fig.10 It can be seen that the lesion-like mutant rbl1 grows dwarfed, and the RBL3 gene is overexpressed in the mutant rbl1 material to obtain the material OERBL3-rr. The growth of OERBL3-rr is consistent with the WT phenotype. It can be seen that expressing the rice RBL3 gene in the rice rbl1 mutant can complement the growth defect of the lesion-like mutant rbl1, that is, there is redundancy between the RBL3 protein and RBL1 functions in rice.

[0122] Example 8

[0123] It can be seen from the literature [Song, X., Meng, X., Guo, H. et al. Targeting a gene regulatory element enhances rice grain yield by decoupling panicle number and size. Nat Biotechnol 40, 1403-1411 (2022). https: / / doi.org / 10.1038 / s41587-022-01281-7] that the determining factors of yield depend on tillering and panicle length. The present invention conducted statistics on agronomic traits such as plant height, tillering, and panicle length of the wild type and the rbl3 mutant. The statistical results are as follows Fig.11 As shown. Fig.11 It can be seen that the growth phenotype of the rbl3 mutant is not affected, and the tillering and ear length are not affected. It can be seen that the yield of the rbl3 mutant is not affected compared with the wild type.

[0124] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. RBL3 The use of genes in 1) and / or 2), RBL3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2: 1) Regulate rice lipid metabolism; 2) Knockout RBL3 The gene improves rice disease resistance and basic active oxygen content; the diseases targeted by the rice disease resistance are rice blast and / or bacterial blight; The regulation of rice lipid metabolism activity is: knocking out RBL3 Genes to increase phosphatidic acid content in rice, knockout RBL3 Genes reduce phosphatidylinositol content in rice, knockout RBL3 Genetic reduction and knockout of phosphatidylserine content in rice RBL3 The gene reduces one or more of the phosphatidylinositol phosphate content in rice.

2. Knockout RBL3 Application of a recombinant vector of a gene in improving rice disease resistance; the rice disease resistance targets rice blast and / or bacterial blight; the knockout RBL3 The recombinant vector of the gene includes a target sequence and a CRISPR / Cas9 vector; the nucleotide sequence of the target sequence is shown in SEQ ID NO.4; RBL3 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. The use according to claim 2, characterized in that: The CRISPR / Cas9 vector includes a pRGEB32 vector.

4. The use according to claim 3, characterized in that: The knockout RBL3 The recombinant vector of the gene includes the sequence shown in SEQ ID NO.23 and a double-enzyme-digested pRGEB32 vector; the enzymes used in the double-enzyme-digestion are FOKI and BSAI.

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