CN118374518B

CN118374518B - Application of RBL3 gene in regulating rice immunity level and/or rice disease resistance

Info

Publication number: CN118374518B
Application number: CN202410487882.2A
Authority: CN
Inventors: 李国田; 韩欣雨; 孔晓静; 郭镇华
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2024-04-22
Filing date: 2024-04-22
Publication date: 2025-03-21
Anticipated expiration: 2044-04-22
Also published as: CN118374518A

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 white leaf 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

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

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of RBL3 genes in regulating and controlling the immune level and/or disease resistance of rice.

Background

The rice blast is one of the most serious fungal diseases which damage rice production, is a common disease in rice fields, and has the characteristics of long damage time, multiple infection positions, multiple symptoms and the like. The rice blast can occur in the whole growth period of the rice, mainly damages the positions of seedlings, leaves, ears, knots and the like, and can form diseases such as seedling blast, leaf blast, knot blast, neck blast, grain blast and the like according to different harmful positions.

At present, rice blast disease prevention and control mainly comprises planting disease-resistant varieties and chemical pesticides. With the perfection of rice genome sequencing, research on rice resistance genes is increasing. Since the 60 s of the 20 th century, researchers have screened rice blast resistance genes Pia, pii and Pik from breeding materials, new rice varieties with excellent resistance such as F you 498, yixiangyou 2115, zhongke 804, longyou 3189 and the like have been cultivated, but with global climate change, agricultural production scale and international grain trade increase, pathogen colony evolution and propagation speed are continuously accelerated, resistance of a single disease-resistant variety is gradually lost after many years of planting, and the introduction of a plurality of R genes (RESISTANCE GENES) is easy to influence grain yield. Therefore, cloning of durable disease-resistant genes, cultivation of high quality materials that are broad-spectrum disease-resistant and do not cause yield loss are of great importance. The R gene is also in the current situation of unclear mechanism analysis and resource shortage. At the same time, the resistance of the R gene fails around 4 years. In addition, fungi are evolving and mutating rapidly during growth. Although a plurality of R genes are superimposed, a longer time and cost are required for cultivating high-quality rice.

Disclosure of Invention

In order to solve the problems, the invention provides application of RBL3 genes in regulating and controlling the immune level and/or disease resistance of rice. The RBL3 gene is identified, and the RBL3 mutant is obtained based on the CRISPR/Cas9 system, so that the mutant has broad-spectrum disease resistance and can help researchers to later screen high-quality disease-resistant materials.

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

The invention provides application of RBL3 genes in one or more of 1) to 4), wherein the amino acid sequence coded by the RBL3 genes is shown as SEQ ID NO. 2:

1) regulating and controlling lipid metabolism activity of rice, 2) regulating and controlling growth of rice, 3) regulating and controlling immune level of rice, and 4) regulating and controlling disease resistance of rice.

Preferably, the regulation of rice disease resistance comprises knocking out RBL3 genes to improve rice disease resistance.

Preferably, the diseases against which the rice disease resistance is directed include rice blast and/or bacterial leaf blight.

Preferably, the regulation of rice lipid metabolism comprises one or more of the steps of knocking out RBL3 genes to increase the phosphatidic acid content of rice, knocking out RBL3 genes to reduce the phosphatidylinositol content of rice, knocking out RBL3 genes to reduce the phosphatidylserine content of rice and knocking out RBL3 genes to reduce the phosphatidylinositol phosphate content of rice.

Preferably, the modulating the immune level of rice comprises knocking out the RBL3 gene to increase the immune level of rice.

The invention provides a recombinant vector for knocking out RBL3 genes, which comprises a target sequence and a CRISPR/Cas9 vector, wherein the nucleotide sequence of the target sequence is shown as SEQ ID NO.4, and the amino acid sequence encoded by the RBL3 genes is shown as SEQ ID NO. 2.

Preferably, the CRISPR/Cas9 vector comprises pRGEB vectors.

Preferably, the recombinant vector for knocking out the RBL3 gene comprises a sequence shown in SEQ ID NO.23 and a pRGEB vector for double enzyme digestion, wherein enzymes used for the double enzyme digestion are FOKI and BSAI.

The invention provides application of the recombinant vector in improving the immunity level and/or the disease resistance of rice.

The beneficial effects are that:

The invention identifies a broad-spectrum disease-resistant gene RBL3, belongs to a member of CDS gene family, codes a CDP-DAG synthetase and regulates rice growth and immunity level by regulating rice lipid metabolism activity. The RBL3 gene in the rice is knocked out by a gene editing technology, so that the rice is found to show obvious broad-spectrum disease resistance to rice blast bacteria and white leaf blight bacteria. In addition, the gene is highly conserved in crops, can be widely popularized in a plurality of species through a means of gene editing, can reduce breeding time, improve crop disease resistance level, yield and quality, breaks species limitation and has stronger universality compared with the traditional over-expression disease resistance gene, has huge disease resistance breeding application potential, and can be widely applied to disease resistance research of a plurality of crops.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a phylogenetic tree of RBL3 homologous proteins of different species;

FIG. 2 shows the result of yeast cds1 back-filling verification of RBL3 function;

FIG. 3 is a schematic representation of a recombinant CRISPR vector of example 3;

FIG. 4 is a schematic diagram of gene editing and a comparative phenotype diagram of rbl3 mutant and Wild Type (WT), wherein a is a schematic diagram of gene editing and b is a comparative phenotype diagram;

FIG. 5 shows basic reactive oxygen species assay results for rbl3 mutants and wild type;

FIG. 6 shows the results of rbl3 mutant and wild type bacterial blight and rice blast;

FIG. 7 shows the results of measurement of the expression levels of rbl3 mutant and wild-type disease-resistance related genes;

FIG. 8 is a graph showing the results of experiments for regulating lipid metabolism activity in rice;

FIG. 9 shows the growth phenotype of rbl1 mutants and the pathogenesis of bacterial blight and Pyricularia oryzae;

FIG. 10 is a graph showing the results of the anaplerotic plaque phenotype of RBL3 over-expression in RBL1 mutants;

FIG. 11 is a statistical result of plant height, tillering and ear length of wild type and rbl3 mutants.

Detailed Description

In the invention, the regulation of rice lipid metabolism activity preferably comprises one or more of the steps of knocking out RBL3 genes to improve the phosphatidic acid content of rice, knocking out RBL3 genes to reduce the phosphatidylinositol content of rice, knocking out RBL3 genes to reduce the phosphatidylserine content of rice and knocking out RBL3 genes to reduce the phosphatidylinositol phosphate content of rice, the regulation of rice growth preferably comprises the steps of expressing RBL3 genes to supplement the growth defect of rice spot mutants, namely that the RBL3 proteins and RBL1 functions in rice are redundant, the regulation of the immunity level of rice preferably comprises the step of knocking out RBL3 genes to improve the immunity level of rice, the regulation of the disease resistance preferably comprises the step of knocking out RBL3 genes to improve the disease resistance of rice, the disease resistance of the rice preferably comprises rice blast and/or bacterial leaf blight, and the coding sequence of the RBL3 genes is preferably shown in SEQ ID NO.1, and the sequence information is as follows:

The RBL3 coding region sequence is shown as SEQ ID NO.1, and is specifically as follows ：5'-ATGCCCCATGTTAGATCCGCCGCTGAAAGGGACAATGGCTCTGGTGGTGATGTCACCCCGGGA ACTCCAAGCCCGACCCATGGTGCCCGTGTGAGGCAGCGGAAGCGCTCCAGCGATGCTCCATCTGACGTGAACAAAACCAATGGCGCTAATTTGCTGCTAAATGACCAGAACAAGTACAAGTCAATGCTTATTCGCACGTATTCTTCGCTCTGGATGATGGCAGGATTTGTGTTTTTGATATACATGGGACATCTATACATATGGGCTATGGTTGTCGTTATCCAAATCTTTATGGCGAGTGAGCTCTTCAATCTGCTGAGGAAGGCCAACGAGGACAGGCAGCTTCCAGGGTTCAGACTACTTAACTGGCATTTCTTCTTCACTGCGATGCTTTTTGCGTATGGCCGTTTCCTTAGTCGGCAGCTTGTCAACACGGTGACTTCAGACAAGTTATTGTACAAGCTTGTGAGTGGCTTGATCAAGTACCAAATGTTTATTTGCTACTTTCTTTATATAGCAGGGTTTGTATGGTTTATTCTCACTCTAAAGAAAAAGGCGTACAAGTATCAATTCAGTCAATATGCATGGACTCACATGATCCTATTGATGGTATTTGCACAGTCATCTTTCACTGTGGCCAATATATATGAAGGAATGTTTTGGTTCCTCCTACCAGCTTCTTTGATCGCTATCAACGATGTTGCTGCATATTTCTTCGGCTTCTTCTTTGGGAAAACACCCTTAATTAAGCTCTCTCCAAAAAAAACTTGGGAGGGCTTTCTAGGTGCATCAGTGACTACCATGCTTTCTGCATTTGTGCTTGCTAATTTCATGGGTCATTTCCAGTGGTTAACATGCCCCAGAAAGGATTTGTCGACTGGATGGCTTCATTGTGATCCTGGCTCTATATTTACACCAGAAAGTTATGATTTACCAGGATGGATCCCTTGGCGAGAAGTGGCAATTATGCCTATTCAGTGGCATGCCTTAGCTCTTGGCTTGTTTGCTTCAATAATAGCACCATTTGGAGGGTTTTTCGCTAGTGGTTTTAAAAGAGCTTTCAAATTCAAGGATTTTGGTGATAGCATACCTGGCCATGGTGGATTTACTGACAGGATGGATTGTCAAATGGTTATGGCTGTGTTTGCTTATATCTATTACCAATCGTTTGTGATGGTACAGGACTTATCTGTTGAGACAATCATGGAGCAGATACTGAGAAATCTCACGTTCGAGGAGCAGCATGATCTTTACGAGCAACTAGGCAAGTTACTGACGAGAGGGAACTGA-3';

The RBL3 protein sequence is shown as SEQ ID NO.2, and is specifically as follows ：MPHVRSAAERDNGSGGDVTPGTPSPTHGARVRQRKRSSDAPSDVNKTNGANLLLNDQNKYKSMLIRTYSSLWMMAGFVFLIYMGHLYIWAMVVVIQIFMASELFNLLRKANEDRQLPGFRLLNWHFFFTAMLFAYGRFLSRQLVNTVTSDKLLYKLVSGLIKYQMFICYFLYIAGFVWFILTLKKKAYKYQFSQYAWTHMILLMVFAQSSFTVANIYEGMFWFLLPASLIAINDVAAYFFGFFFGKTPLIKLSPKKTWEGFLGASVTTMLSAFVLANFMGHFQWLTCPRKDLSTGWLHCDPGSIFTPESYDLPGWIPWREVAIMPIQWHALALGLFASIIAPFGGFFASGFKRAFKFKDFGDSIPGHGGFTDRMDCQMVMAVFAYIYYQSFVMVQDLSVETIMEQILRNLTFEEQHDLYEQLGKLLTRGN.

In the invention, the CRISPR/Cas9 vector preferably comprises pRGEB vector, the recombinant vector for knocking out the RBL3 gene preferably comprises a sequence shown in SEQ ID NO.23 and a double-enzyme-digested pRGEB vector, and enzymes used by the double enzyme digestion are FOKI and BSAI.

The invention provides application of the recombinant vector in improving the immunity level and/or the disease resistance of rice. In the present invention, the diseases against which the rice disease resistance is aimed preferably include rice blast and/or bacterial leaf blight.

For further explanation of the present invention, the application of the RBL3 gene provided in the present invention to regulation of immune levels and/or disease resistance of rice will be described in detail with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

RBL3 protein conservation analysis

In order to explore the biological functions of the RBL3 gene (the coding region sequence is shown as SEQ ID NO. 1), the invention firstly analyzes the sequence specificity of the amino acid sequence (SEQ ID NO. 2) encoded by the RBL3 gene in different biological species. The invention searches all RBL3 homologous proteins in plants, animals and microorganisms through Protein BLAST functions on NCBI, and uses MEGA11 and other sequence analysis tools to compare RBL3 amino acid sequences in different species, and the result is shown in figure 1. The result shows that RBL3 has a conserved CTP-transf _1 structural domain, wherein the amino acid sequence of the RBL protein C-terminal functional structural domain (KDXXXXXPGHGGXXDRXD, SEQ ID No.3, X is any amino acid) is highly conserved in all animals, plants and microorganisms, and can be widely popularized in a plurality of species by means of gene editing so as to accelerate disease-resistant breeding research.

Example 2

In order to further verify whether RBL3 protein in rice has the function of CDP-DAG synthetase (RBL 3), the invention performs function complement verification in Saccharomyces cerevisiae. As a result of the death of Cds1 mutation in yeast, the method described in 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.】 of the invention firstly constructs a galactose-induced RBL3 expression vector, converts a yeast BY4741 strain, screens a Cds1-CoRBL3 positive strain on a YPGal culture medium, and is named Sccds1/OsCDS3. Sccds1 strain 1/OsCDS strain and yeast wild-type strain BY4741 (WT) were cultured on YPGal medium and YPD medium, respectively, and the culture results are shown in FIG. 2.

The galactose-induced RBL3 expression vector was constructed as follows:

The primer pairing mode is that PYES2/RBL3-F and PYES2/RBL3-R are amplified by PCR by taking a Kitaake rice cDNA sequence as a template, and the sequence information is as follows:

PYES2/RBL3-F:5'-ACGATGACGATAAGGTACCTAATGCCCCATGTTAGATCCG-3',SEQ ID NO.24;

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

The reaction conditions of the PCR amplification are 95 ℃ pre-denaturation for 5min, 95 ℃ denaturation for 30s,55 ℃ annealing for 30s,72 ℃ extension for 2min and 32 cycles;

The PCR amplification reaction system comprises ddH ₂ O5.2 mu L, 2X Phanta Mix mu L, dNTP 0.4 mu L, primer F1 mu L, primer R1 mu L, template 2 mu L and Phanta enzyme 0.4 mu L.

The amplified cDNA fragment was ligated with BamHI-digested (cleavage site: GGATCC) pYES2 vector to obtain RBL3 expression vector.

As shown in FIG. 2, in YPD medium, yeast cdsC strain can not grow normally due to the fact that the transferred rice RBL3 gene is not expressed, while in YPGal medium, the added galactose induces the expression of rice RBL3, and yeast can grow normally. Therefore, the expression of the rice RBL3 gene in the yeast cds1 mutant can complement the growth defect of the yeast cds1 mutant, namely, the RBL3 protein in the rice has the function of CDP-DAG synthetase.

Example 3

The CRISPR vector construction method is derived from article 【Xie K,Minkenberg B,Yang Y.Boosting CRISPR/Cas9multiplex editing capability with the endogenous tRNA-processing system[J].National Academy of Sc iences,2015(11).DOI:10.1073/PNAS.1420294112.】. as follows, and the sequences of the primers in the following steps are the same as in the published article unless specifically noted:

(1) Primer design:

the RBL3 gene LOC number LOC_Os10g17990 was first entered into the rice gene database website (http:// www.ricedata.cn/gene /). Finding a proper target point according to RBL3 genes:

The design and editing target sequence is gRNA-30:5'-GCCATTGTCCCTTTCAGCGG-3' (SEQ ID NO. 4), the target is positioned on the second exon of the gene, the off-target rate is low, a gRNA scaffold sequence is connected to the target sequence after the target sequence, namely 5'-GTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC-3', SEQ ID NO.5, and the design primer is designed by taking the target as a reference as follows:

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

RBL 3-R5'-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.

(2) The primer pairing mode is that L5AD5-F and RBL3-R, RBL3-F and L3AD3-R are amplified by PCR by taking a PGTR sequence synthesized artificially as a template, and the sequence information is as follows:

L5AD5-F:5'-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCAACAAAGCACCAGTGG-3',SEQ ID NO.8;

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

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

The reaction conditions of the PCR amplification are 95 ℃ pre-denaturation for 5min, 95 ℃ denaturation for 30s,50 ℃ annealing for 30s,72 ℃ extension for 30s and 32 cycles, and 72 ℃ extension for 5min;

The PCR amplification reaction system comprises ddH ₂ O6.2 mu L, 2X Phanta Mix mu L, dNTP 0.4 mu L, primer F1 mu L, primer R1 mu L, template 1 mu L and Phanta enzyme 0.4 mu L.

(3) GG reaction, wherein PCR products obtained by amplification are purified and then subjected to GG reaction, the reaction system of the GG reaction is that two PCR products are 7 mu L (25-50 ng, the mass ratio is 1:1), 2 xT 4 ligase Buffer (NEB) is 10 mu L, BSA is 2 mu L, bsaI (10U/mu L, NEB) is 0.5 mu L, T4 DNA ligase is 0.5 mu L, and the reaction conditions of the GG reaction are 37 ℃ for 5min,20 ℃ for 10min,40 cycles and 20 ℃ for 1h.

(4) GG PCR, namely adding 180 mu L of double distilled water into the reaction product to dilute, and amplifying a target fragment by using S5AD5-F and S3AD3-R to purify the PCR product, wherein the primer sequence is as follows:

S5AD5-F:5'-CGGGTCTCAGGCAGGATGGGCAGTCTGGGCA-3',SEQ ID NO.11;

S3AD3-R:5'-TAGGTCTCCAAACGGATGAGCGACAGCAAAC-3',SEQ ID NO.12;

The reaction system is the same as the step (2), and the reaction procedure is that the reaction is carried out for 5 minutes at 95 ℃, 30 seconds at 50 ℃, 30 seconds at 72 ℃ for extension, 35 cycles, and 5 minutes at 72 ℃.

(5) And (3) enzyme digestion and enzyme ligation, namely, carrying out enzyme digestion on the fragment obtained by the amplification in the step (4) and pRGEB carrier by using FOKI and BSAI respectively, and reacting for 2 hours at 37 ℃. After the digestion is completed, a digestion fragment containing the RBL3 gene target sequence (the sequence after digestion is shown as SEQ ID NO. 23) and a digestion pRGEB vector are used for carrying out a ligation reaction, and then the escherichia coli Top10 is transformed. Positive clones were screened by PCR (amplification primer pRGEB32/F：5'-aaaagcatttcgtagtgggc-3',SEQ ID NO.26;pRGEB32/R：5'-ttctggctggtttgttggtc-3',SEQ ID NO.27) to obtain recombinant CRISPR vector; the sequence shown in SEQ ID NO.23 is specifically as follows ：5'-GGGTCTCAGGCAGGATGGGCAGTCTGGGCAaacaaagcaccagtggtctagtggtagaatagtaccctgccacggtacaga cccgggttcgattcccggctggtgcaGCCATTGTCCCTTTCAGCGGgttttagagctagaaatagcaagttaaaataaggctagtccgttatc aacttgaaaaagtggcaccgagtcggtgcTTTTTTTTTTGTTTGCTGTCGCTCATCCGTTTGGAGACCTA-3'.

(6) Transformation by combining the recombinant CRISPR vector with the genetic transformation technology mediated by agrobacterium from the Wuhan Boehmeria company to transform rice Kitaake material, and identifying the genotype of the T0 generation gene editing rice material by Sanger sequencing. The T1 generation screening resulted in homozygous mutant material for rbl3 (FIG. 4).

Example 4

Fungal cell walls contain abundant chitin (ptin), and researchers often treat plant tissues with chitin to simulate pathogen-associated molecular pattern-stimulated immune response (PTI) and to initially determine plant disease resistance levels based on the extent of reactive oxygen species outbreaks. The invention uses chitin to treat wild and mutant seedling stage leaves, and uses a chemiluminescence method to observe active oxygen burst, and the specific method is as follows:

Culturing seeds of wild rice Kitaake and mutant rice rbl3 on a 1/2MS sterile medium, cutting seedling leaves into small pieces (3 mm multiplied by 3 mm) after 12 days, placing the cut leaves into 96-well plates, adding 100 mu L of sterile double distilled water into each well for resuscitating for 8-10 hours, sucking out resuscitating solution, adding 100 mu L of reaction solution containing 50 mu M/L Luminol (Luminol), 10 mu g/mL peroxidase (HRP) and 8nM chitin, and immediately starting measurement, and measuring chemiluminescence at 500MS intervals within 40 minutes. Each sample was set up with 3 biological replicates, each replicate containing 8 technical replicates, with sterile double distilled water treatment as negative control (mock). The results are shown in FIG. 5.

As can be seen from fig. 5, the rbl3 mutant had significantly higher basal reactive oxygen species than the wild type, and the reactive oxygen species of the mutant leaves accumulated rapidly after chitin treatment.

Example 5

Because the mutant is extremely easy to trigger apoptosis by itself, pathogenic bacteria are difficult to expand infection in the body, and generally have higher disease resistance. The invention carries out disease resistance identification on the rbl3 mutant inoculated bacterial leaf blight bacteria and rice blast bacteria. The method comprises the following steps:

identification of rice blast resistance

(1) Culture of Pyricularia oryzae

Inoculating paper sheet containing Pyricularia oryzae (ZB 25) spores onto oat culture medium, dark culturing at about 25deg.C for 2-3 days, culturing under illumination after hypha germinates, scraping hypha (containing a large number of conidia) with distilled water containing 0.01% Tween-20 within two weeks when Pyricularia oryzae grows over the whole culture dish, filtering with double-layer gauze, calculating spore concentration with microscope and blood cell counting plate, regulating spore suspension concentration to 2× ⁵ spores/mL for Pyricularia oryzae inoculation, wherein Pyricularia oryzae (ZB 25) is derived from agricultural university in China, and is disclosed in 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.】.

(2) Inoculation method

In the four-leaf stage of rice (rbl 3 mutant or wild type), a potential circular wound was artificially made on the young plant newly-extended leaf by using a mouse ear punch on the inverted two-leaf, 10. Mu.L of the bacterial droplets were sucked up at the wound by using a pipette and blocked by using transparent adhesive tape, 5 strains were inoculated for each strain, 3 independent inoculations were repeated, and then the strain was transferred to 28 ℃ high humidity condition for further growth, and the disease condition (phenotype and disease area) was investigated after 14 days of inoculation. The result is shown as a in FIG. 6, scale 1cm.

Identification of bacterial leaf blight resistance

1) Culture of bacterial leaf blight bacteria

The bacterial leaf blight bacteria (PXO 99) are cultured by using NB culture medium at 28 ℃ for 2-3 days, when the bacterial leaf blight bacteria grow on the whole culture dish, the bacterial cells are scraped out, and after the bacterial leaf blight bacteria grow on the whole culture dish, ddH ₂ O is obtained after sterilization, and the bacterial leaf blight bacteria are uniformly mixed until the OD is 0.5, and then the bacterial leaf blight bacteria are inoculated. The bacterial leaf blight bacteria (PXO 99) are derived from the university of agricultural in China and are 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.】.

2) Inoculation method

The prepared bacterial liquid is dipped on the leaves of the rice (rbl 3 mutant or wild type) in the 4-leaf stage and the 7-leaf stage by a leaf shearing method, each strain is inoculated with 5 strains, the inoculation is repeated for 3 times, and the disease spot length and the disease leaf length are investigated after 14 days of inoculation, so that the disease condition is investigated. The result is shown in FIG. 6b, scale bar 1cm.

As can be seen from FIG. 6, after 14 days of inoculation, bacterial leaf blight bacteria only expand by 7cm on the mutant leaves, which is significantly lower than that of the wild type 14cm, and the area of rice blast bacterial plaque is significantly lower than that of the wild type.

Example 6

Identification of rbl3 mutant disease resistance gene expression level

In the process of co-evolution of plants and pathogenic bacteria for a long time, various immune regulation modes are established to control the expression of PR and other disease-resistant genes. The invention takes RNA of 4-week-old rice (rbl 3 mutant or wild type) leaves for analysis, takes action as an internal reference gene for qRT-PCR analysis, and has the following primer sequences:

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

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

qRT-PR1aF:5'-CGTGTCGGCGTGGGTGT-3',SEQ ID NO.15;

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

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

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

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

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

the qRT-PCR reaction system is that ：2×ChamQ SYBR qPCRMaster Mix 10μL,F-primer(10μM)0.4μL,R-primer(10μM)0.4μL,cDNA 2μL,ddH₂O 7.2μL.

The qRT-PCR reaction program is 95 ℃ pre-denaturation for 30s, 95 ℃ denaturation for 10s,60 ℃ annealing for 30s and 40 cycles;

As a result, it was found that the expression of the immune-related genes in rbl3 mutant leaves was significantly upregulated, several times higher than that of the wild type, without infection by pathogenic bacteria (FIG. 8). By analyzing the phenotype of rbl3 mutant and rice disease resistance related factors, it is verified that the mutant gene plays an important role in the immunization, growth and development of rice.

Example 7

1. Regulating lipid metabolism activity of rice

In order to analyze the phospholipid metabolic process regulated by RBL3 protein, the invention analyzes the change condition of phospholipid metabolism in the RBL3 mutant, and the experimental process is as follows:

Cutting 5-week-old rice leaves into 3cm small pieces, soaking in 3mL isopropanol (containing 0.01% BHT), placing in 75 ℃ water bath for 15 min, adding 3mL chloroform/methanol (2:1) solution (containing 0.01% BHT), oscillating for 1 hr, collecting extractive solution, repeatedly adding new chloroform methanol mixture, and extracting until all leaves turn white. 2mLKCl (1M) solution was added to the extraction solution, and after vortexing, the mixture was centrifuged at 4℃and 12,000Xg to remove the aqueous phase.

The lower organic phase was dried with nitrogen and redissolved in chloroform (10 mg/mL). The lipidomic analysis method was performed 【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.】, with reference to the steps in the published literature using a TripleTOF mass spectrometer (TripleTOF-MS/MS) in positive ion mode, ionization data were collected and relative quantitative analysis was performed by the internal standard method (Avanti).

The results are shown in FIG. 8. The results showed that the RBL3 mutant showed a 2-fold increase in Phosphatidic Acid (PA) compared to wild type and a significant decrease in Phosphatidylinositol (PI), phosphatidylserine (PS) and phosphatidylinositol phosphate (PIP), which indicated that RBL3 protein was involved in phospholipid metabolism.

2. Regulating rice growth:

According to the invention, a disease-like mutant rbl1 is screened from a rice Kitaake fast neutron mutant library, and the resistance of the mutant to fungal disease rice blast and bacterial leaf blight is enhanced. However, growth was severely affected and grown very short, as shown in FIG. 9, FIG. 9 was derived from literature 【Sha G,Sun P,Kong X,et al.Genome editing ofa rice CDP-DAG synthase confers multipathogen resistance[J].Nature,2023,618(7967):1017-1023.】, wherein a, growth phenotypes of wild type Kitaake X (WT) and rbl1 mutants were 40 days after greenhouse sowing, scale bar, 10cm; b, leaf phenotype of wild type and mutant in the adult stage, scale bar, 1cm; c, bacterial blight inoculated wild type and mutant flag leaf 14 day phenotype, d, bacterial blight inoculated disease length statistics of each strain after 14 days, e, rice blast puncture inoculated wild type and mutant leaf 14 day phenotype, f, bacterial blast inoculated for 14 days disease area (left) and fungal biomass (right) statistics, rice blast and bacterial blight resistance identification experimental procedure was detailed in example 5.

The method described in the reference 【Sha G,Sun P,Kong X,et al.Genome editing ofa rice CDP-DAG synthase confers multipathogen resistance[J].Nature,2023,618(7967):1017-1023.】 of the invention firstly constructs an RBL3 over-expression vector, and entrusts the Wuhan Boer remote organism company to transform rice mutant RBL1 material by using an agrobacterium-mediated genetic transformation technology, and the material is named OERBL-rr. OERBL3-rr results are shown in FIG. 10.

The full-length genome sequence of the RBL3 gene is cloned into a stable transgenic expression pRGV vector containing a corn Ubiquitin10 promoter, and the RBL3 overexpression vector is constructed as follows:

The primer pairing mode is that the OE-RBL3/F and the OE-RBL3/F are subjected to PCR amplification by taking a Kitaake rice DNA sequence as a template, and the sequence information is as follows:

OE-RBL3/F:5'-gatatccagatccagtgGGATCCATGCCCCATGTTAGATCCG-3',SEQ ID NO.28;

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

And (3) carrying out a ligation reaction on the amplified DNA fragment and a pRGV vector subjected to Kpn1I digestion (digestion site: GGTACC) to obtain an RBL3 over-expression vector.

As can be seen from FIG. 10, the plaque-like mutant RBL1 grew short and the RBL3 gene was overexpressed in the mutant RBL1 material to obtain material OERBL-rr. OERBL3-rr growth was consistent with the WT phenotype. Therefore, the expression of the rice RBL3 gene in the rice RBL1 mutant can complement the growth defect of the disease spot mutant RBL1, namely, the RBL3 protein in the rice and the RBL1 function have redundancy.

Example 8

It is known from 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 yield determinant depends on tillering and ear length. The invention performs statistics on agronomic characters such as plant height, tillering, spike length and the like of wild type and rbl3 mutants, and the statistical results are shown in figure 11. From fig. 11, rbl3 mutant growth phenotype was not affected, tillering and ear length were not affected, and it was seen that rbl3 mutant yield was not affected relative to wild type.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. Use of the RBL3 gene in 1) and/or 2), wherein the amino acid sequence encoded by the RBL3 gene is shown in SEQ ID NO.2:

1) Regulating rice lipid metabolism activities; 2) Knocking out the RBL3 gene to improve rice disease resistance and basal reactive oxygen content; the rice disease resistance is directed against rice blast and/or bacterial leaf blight;

The regulation of rice lipid metabolism activity is: 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.

2. Application of a recombinant vector for knocking out the RBL3 gene in improving rice disease resistance; the diseases targeted by the rice disease resistance are rice blast and/or bacterial blight; the recombinant vector for knocking out the RBL3 gene comprises a target sequence and a CRISPR/Cas9 vector; the nucleotide sequence of the target sequence is shown in SEQ ID NO.4; the amino acid sequence encoded by the RBL3 gene is shown in SEQ ID NO.2.

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

4. The use according to claim 3, characterized in that 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.