Rice immune negative regulatory protein OsPIS and its mutants and applications

Abstract

The present invention discloses a rice immune negative regulatory protein, OsPIS, and its mutants and applications, belonging to the field of plant genetic engineering technology. The OsPIS knockout mutant plants, when inoculated with the rice blast fungus, affect the attachment and germination of the pathogen's conidia, activating immune responses in rice, including programmed cell death, reactive oxygen species (ROS) production, callose deposition, and expression of PR defense response genes. This significantly improves rice resistance to blast, and can be effectively applied to molecular breeding for disease resistance in rice, with broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a rice immune negative regulatory protein OsPIS and a mutant and application thereof. Background Art

[0002] During their growth and development, plants are often attacked by a variety of pathogens, including fungi, bacteria, viruses, and nematodes. Rice blast is one of the most serious fungal diseases affecting rice production, resulting in annual grain losses of 10%-30%. Therefore, improving plant disease resistance is crucial for effectively controlling crop diseases.

[0003] Phosphatidylinositol (PI) is one of the major phospholipids in eukaryotic cells. It is synthesized from cytidine diphosphate diacylglycerol (CDP-DAG) and free myo-inositol (myo-inosito1) by phosphatidylinositol synthase (PIS) and distributed to other subcellular locations through lipid transfer proteins, targeted vesicular transport, or a combination of both (Phillips et al., 2006). PI is a key membrane component and a precursor of inositol-containing lipids found in all plants and animals (Balla et al., 2013), essential for the growth and metabolism of organisms ( et al., 1993). For example, by modulating the PI system, it is possible to generate plants with altered sensitivity to environmental stresses such as drought. In addition to their structural roles, phospholipids can serve as essential cofactors for membrane enzymes, or they can act as signal precursors or actual signaling molecules (Martin et al., 1998; Meijer et al., 2003). Over the past few years, membrane-associated phospholipids have attracted considerable attention in the study of plant signal transduction.

[0004] PIS, a key enzyme in PI synthesis, has been extensively studied in yeast. Di et al. purified and isolated PIS from yeast cytoplasm and, through functional analysis, found it to be present in nearly all cell types, localized to the endoplasmic reticulum, Golgi apparatus, and mitochondrial outer compartment (Di and De, 2006). PIS activity and localization were first discovered in guinea pig tissues by Benjamins and Agranoff. They detected PIS activity in organs such as brain, kidney, and lung, and found that the synthase expressed differently in different tissues (Benjamins et al., 1969). As for plants, relatively little is known about PIS. PIS genes have only been cloned from a few plants, including maize, Arabidopsis thaliana, Brassica napus, and rice, and reports on protein purification and characterization are few and far between. Current research suggests that phosphatidylinositol synthases are involved in plant responses to stresses such as salt and drought. Das et al. cloned the BnPIS1 gene from Brassica napus. BnPIS1 transcription levels vary across different tissues and developmental stages in Brassica napus, and it responds to environmental conditions such as high salt, low temperature, and drought (Das et al., 2005). Zhang et al., after introducing the ZmPIS promoter into tobacco, increased its response to NaCl or PEG stress (Zhang et al., 2016). However, whether PIS participates in disease defense responses remains unknown. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a rice immune negative regulatory protein, OsPIS, and its mutants and applications. The present invention discovered that a rice phosphatidylinositol synthase-deficient mutant (OsPIS-ED) improves rice resistance to rice blast by regulating PTI-related reactive oxygen species (ROS) bursts, callose deposition, and activation of defense gene expression.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of rice immune negative regulatory protein OsPIS in improving rice disease resistance;

[0008] The rice immune negative regulatory protein OsPIS is a protein shown in any one of the following (A1) or (A2):

[0009] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO. 2; or

[0010] (A2) Derivative proteins having equivalent functions are obtained by substituting, deleting or adding one or more amino acids to the sequence shown in SEQ ID NO. 2.

[0011] The disease resistance is resistance to rice blast, which is a rice blast disease caused by Magnaporthe oryzae.

[0012] The second aspect of the present invention provides the use of a gene encoding a rice negative immune regulatory protein OsPIS in the following (1) or (2):

[0013] (1) Improve rice disease resistance;

[0014] (2) Cultivate rice varieties with improved disease resistance.

[0015] In the above application, the rice immune negative regulatory protein OsPIS is encoded by the gene Ospis. Preferably, the gene Ospis is a nucleic acid as described in any one of a) or b) below:

[0016] a) a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO.1;

[0017] b) A nucleic acid molecule that has 90% or more identity with the nucleotide sequence of a) and expresses a protein with the same function.

[0018] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated using computer software, for example, the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0019] In the above nucleic acid molecules, the 90% or greater identity may be at least 90%, 91%, 92%, 95%, 96%, 98% or 99% identity.

[0020] In the above application, the disease resistance is resistance to rice blast, which is a rice blast disease caused by Magnaporthe oryzae.

[0021] The third aspect of the present invention provides the use of the following (1) or (2) in improving disease resistance of rice:

[0022] (1) gRNA sequence, recombinant knockout vector or transformant for specific knockout of the Ospis gene;

[0023] (2) A silencing fragment, recombinant silencing vector or transformant for specifically silencing the Ospis gene.

[0024] The disease resistance is resistance to rice blast, which is a rice blast disease caused by Magnaporthe oryzae.

[0025] A fourth aspect of the present invention provides a method for improving disease resistance of rice, comprising:

[0026] Steps to knock out or silence the gene Ospis in rice;

[0027] Alternatively, the steps to delete the function of OsPIS, a negative regulatory protein of immunity in rice.

[0028] Among the above methods, CRISPR-Cas9 gene editing technology, VIGS technology, T-DNA insertion or RNA interference technology can be used to knock out or silence the gene Ospis in rice.

[0029] In a fifth aspect, the present invention provides a mutant of the rice immune negative regulatory protein OsPIS, wherein the amino acid sequence of the mutant is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0030] The sixth aspect of the present invention provides the use of the above mutant in improving the disease resistance of rice.

[0031] The disease resistance is resistance to rice blast, which is a rice blast disease caused by Magnaporthe oryzae.

[0032] Beneficial effects of the present invention:

[0033] The OsPIS knockout mutant plant, described in the present invention, affects the attachment and germination of pathogen conidia after inoculation with rice blast fungus, activates rice immune responses such as programmed cell death, reactive oxygen species burst, callose deposition, and PR defense response gene expression, thereby greatly improving rice resistance to rice blast. The plant can be effectively applied to molecular breeding for disease resistance in rice and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Screening and Identification of OsPIS-ED Mutants (A) Electrophoresis of gDNA PCR products from OsPIS-ED#24 and OsPIS-ED#34 mutants. (B) Sequencing results and alignment of OsPIS-ED#24 and OsPIS-ED#34 mutants. In the figure, OsPIS-ED#24-1, 24-2, and 24-3 represent different strains of the OsPIS-ED#24 knockout line; OsPIS-ED#34-1, 34-2, and 34-3 represent different strains of the OsPIS-ED#34 knockout line.

[0035] Figure 2 :Pathogenesis of OsPIS-ED mutant and wild-type rice after inoculation with rice blast fungus

[0036] (A) Disease status of rice leaves. (B) Statistics of diseased area on rice leaves.

[0037] Figure 3 :Attachment and germination of pathogen conidia on OsPIS-ED mutant and wild-type rice leaves

[0038] (A) The amount of spore attachment at different time periods. (B) The amount of spore germination at different time periods.

[0039] Figure 4 : Trypan blue staining results

[0040] (A) Necrosis of OsPIS-ED mutant and wild-type rice. (B) Necrosis statistics of OsPIS-ED mutant and wild-type rice.

[0041] Figure 5 : Active oxygen burst detection results

[0042] (A) Reactive oxygen species burst in OsPIS-ED mutant and wild-type rice. (B) H2O2 content statistics in OsPIS-ED mutant and wild-type rice.

[0043] Figure 6 : Callose deposition results

[0044] (A) Callose deposition in OsPIS-ED mutant and wild-type rice. (B) Callose count in OsPIS-ED mutant and wild-type rice.

[0045] Figure 7 :Analysis of defense response gene expression

[0046] (A) OsPR1a gene expression level. (B) OsPR10 gene expression level. (C) OsPR5 gene expression level. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0048] As mentioned above, current research has found that phosphatidylinositol synthase is involved in plant responses to stresses such as salt and drought. However, whether PIS is involved in disease resistance has not been reported.

[0049] Based on this, the present invention conducted an in-depth study on the relationship between OsPIS and rice blast resistance. OsPIS is encoded by the gene Ospis, the nucleotide sequence of which is shown in SEQ ID NO. 1 and is as follows:

[0050] ATGGCACAACCTTCTTCTAAGAAGACGCCGTCAGTCTATCTTTACATCCCTAATATCATTGGATATTTTAGGATCATCATAAACTTCATTGCTTTTGCCGTATGTTATTCCAATAGGGTGCCTTTGCTATCCTATACTTCTTCAGCTTTTTCTGCGATGGCTTGGA TGGTTGGTTTGCACGAAAGTTTAACCAAGCATCAACATTTGGAGCTGTGCTGGACATGGTAACAGATAGGGTTAGCACTGCCTGTTTGTTGGCACTTCTCTCCAAGTTTTACAGACCTGGCTTAGTTTTCTTGATGTTGCTTGGGTTGGATATTACAAGCCATTGGT TTCAAATGTACAGTTCGTTCCTATCAGGTAAGACTAGCCACAAGGATGTAAAAGACACAGGCAATTGGCTTCTGAAGTTATATTATGGACACCGACCATTCATGGCCTTCTGTTGTGTTGCTTCGGAGGTTCTGTACATAGTTCTTTTTCTGTTCGCCGATGAGAAG TCAACAAGCTTGCTTAATGTGTGCGGAAACCTTCTGAAGCAAAGTCCTCTCACTGTCTTTGTTTTCATTTCAACTCTAGTTGGTTGGGCGTTGAAACAAGTGATCCAAGTTATCCAGATGAAATCAGCCGCAGACGCGTGCGTTGTGTTTGATTTGAAGCGCGGCAAG

[0051] The amino acid sequence of the OsPIS protein is shown in SEQ ID NO. 2, and is as follows:

[0052] MAQPSSKKTPSVYLYIPNIIGYFRIIINFIAFAVCYSNRVLFAILYFFSFFCDGLDGWFARKFNQASTFGAVLDMVTDRVSTACLLALLSQFYRPGLVFLMLLGLDITSHWF QMYSSFLSGKTSHKDVKDTGNWLLKLYYGHRPFMAFCCVASEVLYIVLFLFADEKSTSLLNVCGNLLKQSPLTVFVFISTLVGWALKQVINVIQMKSAADACVVFDLKRGK.

[0053] The present invention constructs a rice OsPIS gene knockout mutant, inoculates the rice blast fungus strain and compares it with the wild type. The results show that the OsPIS protein has a negative regulatory effect on rice disease resistance and can be effectively applied to molecular improvement breeding of rice disease resistance, thus proposing the present invention.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0055] The experimental materials used in the examples of the present invention are all conventional experimental materials in the field and can be purchased through commercial channels. The present invention introduces the expression vector into the plant cells. The introduction methods are all well known to those skilled in the art, including but not limited to: Agrobacterium-mediated transformation, gene gun method, electroporation method, ovary injection method, etc. If the specific experimental conditions and methods are not specified in the examples of the present invention, they are generally in accordance with conventional conditions, such as J. Sambrook et al., ed., Molecular Cloning Experiment Manual (3rd Edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Manual, Science Press, 2001; or according to the conditions recommended by the manufacturer.

[0056] The public can obtain the rice blast fungus strain Guy11 used in the present invention from the applicant within 20 years from the date of application for use in repeating the experiment.

[0057] Example 1: Screening and identification of OsPIS-ED mutants

[0058] OsPIS-ED knockout mutant plants were constructed using CRISPR / Cas9 gene editing technology using the pYLCRISPR / Cas9Pubi-H vector (stored in our laboratory). http: / / skl.scau.edu.cn / ), proceeded to target design, and determined the gRNA sequence targeting the OsPIS gene: TTTTGCCGTATGTTATTCCAATAGG. The sgRNA expression cassette was constructed using overlapping PCR. In the first round of PCR, the target sequence was introduced downstream of the U3 / U6 promoter and upstream of the sgRNA sequence. A second round of PCR constructed the complete expression cassette containing the promoter, target, and sgRNA. The sgRNA expression cassette was then assembled into the pYLCRISPR / Cas9 vector using the "cut-and-ligate" method, using Bsa I digestion and ligation.

[0059] The correct expression vector pYLCRISPR / Cas9Pubi-H-OsPIS was transformed into Agrobacterium EHA105, and rice Nipponbare was used as the recipient for Agrobacterium transformation. After screening, differentiation, and regeneration, T0 generation transgenic plants were obtained. T0 generation plants were planted individually, and genomic DNA was extracted from leaves of each plant. Primers were designed near the target site and PCR detection was performed. The detection primers PIS-F1 and PIS-R1 are shown in Table 1. The PCR products were sequenced and two mutant strains, OsPIS-ED#24 and OsPIS-ED#34, were obtained. The results are shown in Figure 1. Figure 1 As shown in A.

[0060] The amino acid sequence of the OsPIS protein in the OsPIS-ED#24 mutant strain is shown in SEQ ID NO. 3 and is as follows:

[0061] MAQPSSKKTPSVYLYIPNIIGYFRIIINFIAFAVCFQ*

[0062] The amino acid sequence of the OsPIS protein in the OsPIS-ED#34 mutant strain is shown in SEQ ID NO. 4 and is as follows:

[0063] MAQPSSKKTPSVYLYIPNIIGYFRIIINFIAFAVCLGCSLLSYTSSAFSAMAWMVGLHESLTKHQHLELCWTW*

[0064] *Indicates that the deletion of nucleotides causes the translated protein to terminate prematurely after frameshift.

[0065] The T0 generation homozygous plants were propagated and planted to obtain T1 generation mutant rice. Genomic gDNA from the leaves of the T1 generation mutant plants was extracted and screened and identified using PCR technology. The identification results were sequenced using SnapGene, and finally two stable mutant lines, OsPIS-ED#24 and OsPIS-ED#34, were obtained. Figure 1As shown in B.

[0066] Table 1: Primers for PCR identification of mutant plants

[0067]

[0068] Example 2: Incidence of rice blast inoculated with wild-type and mutant rice

[0069] The rice seedlings grown for 15 days (at least 90% or fully expanded) were inoculated with rice blast fungus. 10 mL of the solution was filled with 1×10 5 Rice strains of OsPIS-ED#24 and OsPIS-ED#34 mutants, as well as wild-type Nipponbare rice, were spray-inoculated with a spore suspension of the rice blast fungus strain Guy11 at a concentration of 10 cfu / mL. An equal amount of water was used as a control. After treatment, the rice plants were shaded and moisturized for 24 hours. Rice growth and disease status were observed daily after inoculation, and disease incidence was counted on the sixth day. After the shading and moisturizing treatment, all rice leaves were kept moist. Three days after inoculation, small lesions were observed on the rice leaf surface. These lesions gradually expanded, and by around the fifth day, all plants were susceptible, with most turning yellow. Diseased leaves were removed on the seventh day for resistance analysis.

[0070] The results are as follows Figure 2 As shown, the results showed that compared with the wild type, OsPIS-ED#24 and OsPIS-ED#34 had significantly fewer lesions on their leaves. The Image J software was used to calculate the diseased area between the two within the same unit leaf area. From the statistical results, the diseased area of ​​the wild type was significantly higher than that of the mutant strain. The above field experiments showed that OsPIS-ED had a negative regulatory effect on rice disease resistance.

[0071] Example 3: Adhesion and germination of conidia of rice blast pathogen on wild-type and mutant rice

[0072] Fresh leaves of the mutants (OsPIS-ED#24, OsPIS-ED#34) and wild-type rice after inoculation with the rice blast fungus Guy11 were cut at 6h, 12h, 24h, and 48h, respectively. The spore attachment and germination on the leaves were observed under an inverted fluorescence microscope and photographed. The images were counted using the counting function of Photoshop to count the number of spore attachment and germinated spores of the mutants and wild-type Nipponbare, respectively, and the average was taken for data analysis.

[0073] The results are as follows Figure 3Statistical results show that conidia aggregated in large numbers during the initial infection phase, with little germination. From 12 hours on, conidia gradually germinated and produced hyphae, increasing over time until germination gradually decreased and stabilized at 48 hours. However, throughout the germination process, the germination rate of wild-type Nipponbare was consistently higher than that of the mutant. This demonstrates that OsPIS-ED affects the germination rate of conidia on rice leaves.

[0074] Example 4: Detection of necrotic cells in rice leaves after inoculation with rice blast fungus by trypan blue staining

[0075] After inoculating OsPIS-ED#24 and OsPIS-ED#34 mutant plants and wild-type Nipponbare with rice blast fungi, new leaves were stained with trypan blue solution. After staining, the leaves were analyzed for cellular necrosis. Based on these experimental results, the area of ​​cellular necrosis per unit leaf area was calculated using Image J software.

[0076] The results are as follows Figure 4 The staining results showed that the degree of cell necrosis in the mutant OsPIS-ED#24 and #34 plants was significantly higher than that in the wild type under the same leaf area, while the cell necrosis in the mutant and wild type lines was less under H2O treatment.

[0077] Example 5: H2O2 detection in rice leaves after inoculation with rice blast fungus

[0078] Ten- to 15-day-old OsPIS-ED#24 and OsPIS-ED#34 mutants and wild-type plants were inoculated with the rice blast pathogen Guy11 and treated with H2O as a control. 24 hours later, 5-8 leaves from each plant were excised and placed in a 50-mL centrifuge tube containing 0.5 mg / mL DAB solution. Vacuum was applied for 1-1.5 hours to completely immerse the leaves in the DAB solution. The tubes were incubated overnight at room temperature and decolorized with 95% ethanol at 37°C until the leaves turned white. The tubes were then photographed using a stereomicroscope. The area of ​​H2O2 per unit leaf area was calculated using Image J software.

[0079] The results are as follows Figure 5 As shown in the figure, under the same area, the mutant plant has a larger ROS burst area than the WT plant.

[0080] Example 6: Detection of callose deposition in rice leaves after inoculation with rice blast fungus

[0081] After spraying a spore suspension of the rice blast pathogen Guy11 and an equal amount of HO, OsPIS-ED#24 and OsPIS-ED#34 mutants and the wild-type Nipponbare were treated, new leaves were stained with 0.1% aniline blue solution to analyze callose deposition. The experiment was repeated three times. The staining results showed that the mutants OsPIS-ED#24 and OsPIS-ED#34 accumulated significant amounts of callose, while the wild-type had significantly lower callose deposition than the mutants. Both mutants and the wild-type showed trace amounts of callose deposition after HO treatment.

[0082] Photoshop software was used to count callose deposition. Compared with the wild-type Nipponbare, the mutant strain deposited more callose per equivalent leaf area. This suggests that OsPIS-ED positively regulates callose deposition in rice leaves during the PTI response.

[0083] Example 7: Detection of defense response genes in rice leaves after inoculation with rice blast fungus

[0084] In this study, RNA was extracted from rice tissues of mutant and wild-type Nipponbare plants at 0, 6, 12, 24, and 48 hours after inoculation with the rice blast pathogen. Real-time quantitative PCR was used to measure the relative expression levels of OsPR1a, OsPR10, and OsPR5 in leaves, using OsActin1 as a reference gene and OsPR1a as the pathogenesis-associated proteins.

[0085] The results are as follows Figure 7 As shown in Figure 2 . Following infection with the rice blast pathogen, OsPIS-ED#24 and #34 plants induced significant upregulation in the expression of the OsPR1a, OsPR10, and OsPR5 genes, reaching a peak at 12 hours and then gradually decreasing over time. In wild-type plants, expression levels peaked at 6 hours, all higher than in the mutant lines, and then gradually decreased at 12, 24, and 48 hours, all lower than in the mutant lines. These results suggest that OsPIS-ED acts as a regulatory factor to activate the expression of plant defense response genes.

[0086] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application. SEQUENCE LISTING <110> Shandong Agricultural University <120> Rice immune negative regulatory protein OsPIS and its mutants and applications <130> 2022 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 669 <212> DNA <213> Rice (Oryza sativa L.) <400> 1 atggcacaac cttcttctaa gaagacgccg tcagtctatc tttacatccc taatatcatt 60 ggatatttta ggatcatcat aaacttcatt gcttttgccg tatgttatattc caatagggtg 120 ctctttgcta tcctatactt cttcagcttt ttctgcgatg gcttggatgg ttggtttgca 180 cgaaagttta accaagcatc aacatttgga gctgtgctgg acatggtaac agatagggtt 240 agcactgcct gtttgttggc acttctctcc cagttttaca gacctggctt agttttcttg 300 atgttgcttg ggttggatat tacaagccat tggtttcaaa tgtacagttc gttcctatca 360 ggtaagacta gccacaagga tgtaaaagac acaggcaatt ggcttctgaa gttatattat 420 ggacaccgac cattcatggc cttctgttgt gttgcttcgg aggttctgta catagttctt 480 tttctgttcg ccgatgagaa gtcaacaagc ttgcttaatg tgtgcggaaa ccttctgaag 540 caaagtcctc tcactgtctt tgttttcatt tcaactctag ttggttgggc gttgaaacaa 600 gtgatcaatg ttatccagat gaaatcagcc gcagacgcgt gcgttgtgtt tgatttgaag 660 cgcggcaag 669 <210> 2 <211> 223 <212> PRT <213> Rice (Oryza sativa L) <400> 2 Met Ala Gln Pro Ser Ser Lys Lys Thr Pro Ser Val Tyr Leu Tyr Ile 1 5 10 15 Pro Asn Ile Ile Gly Tyr Phe Arg Ile Ile Ile Asn Phe Ile Ala Phe 20 25 30 Ala Val Cys Tyr Ser Asn Arg Val Leu Phe Ala Ile Leu Tyr Phe Phe 35 40 45 Ser Phe Phe Cys Asp Gly Leu Asp Gly Trp Phe Ala Arg Lys Phe Asn 50 55 60 Gln Ala Ser Thr Phe Gly Ala Val Leu Asp Met Val Thr Asp Arg Val 65 70 75 80 Ser Thr Ala Cys Leu Leu Ala Leu Leu Ser Gln Phe Tyr Arg Pro Gly 85 90 95 Leu Val Phe Leu Met Leu Leu Gly Leu Asp Ile Thr Ser His Trp Phe 100 105 110 Gln Met Tyr Ser Ser Phe Leu Ser Gly Lys Thr Ser His Lys Asp Val 115 120 125 Lys Asp Thr Gly Asn Trp Leu Lys Leu Tyr Gly His Arg Pro 130 135 140 Phe Met Ala Phe Cys Cys Val Ala Ser Glu Val Leu Tyr Ile Val Leu 145 150 155 160 Phe Leu Phe Ala Asp Glu Lys Ser Thr Ser Leu Leu Asn Val Cys Gly 165 170 175 Asn Leu Leu Lys Gln Ser Pro Leu Thr Val Phe Val Phe Ile Ser Thr 180 185 190 Leu Will Gly Trp Only Leu Lys Gln Will Ile Asn Will Ile Gln Met Lys 195 200 205 Ser Ala Ala Asp Ala Cys Val Val Phe Asp Leu Lys Arg Gly Lys 210 215 220 <210> 3 <211> 37 <212> PRT <213> OsPIS-ED#24 Registered <400> 3 Met Ala Gln Pro Ser Ser Lys Lys Thr Pro Ser Val Tyr Leu Tyr Ile 1 5 10 15 Pro Asn Goes To Gly Tyr Phe Arg Goes To Go To Asn Goes To Ala Phe 20 25 30 Ala Val Cys Phe Gln 35 <210> 4 <211> 73 <212> PRT <213> OsPIS-ED#34 mutant strain <400> 4 Met Ala Gln Pro Ser Ser Lys Lys Thr Pro Ser Val Tyr Leu Tyr Ile 1 5 10 15 Pro Asn Ile Ile Gly Tyr Phe Arg Ile Ile Ile Asn Phe Ile Ala Phe 20 25 30 Ala Val Cys Leu Gly Cys Ser Leu Leu Ser Tyr Thr Ser Ser Ala Phe 35 40 45 Ser Ala Met Ala Trp Met Val Gly Leu His Glu Ser Leu Thr Lys His 50 55 60 Gln His Leu Glu Leu Cys Trp Thr Trp 65 70 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> 5 tggtgagctt ctgtacgatt tct 23 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <400> 6 tgttgagcag tatttcaaca atagc 25

Claims

1. Application of functional deletion of rice immune negative regulatory protein OsPIS in improving rice disease resistance; The amino acid sequence of the rice immune negative regulatory protein OsPIS is shown in SEQ ID NO. 2; The disease resistance is resistance to rice blast, which is caused by the blast fungus ( Magnaporthe oryzae ) causes rice blast.

2. Use of knocking out or silencing the gene encoding the rice immune negative regulatory protein OsPIS according to claim 1 in the following (1) or (2): (1) Improve rice disease resistance; (2) Cultivate rice varieties with improved disease resistance; Rice immune negative regulatory protein OsPIS is expressed by the gene Ospis Encoding, the gene Ospis The nucleotide sequence is shown in SEQ ID NO.1; The disease resistance is resistance to rice blast, which is caused by the blast fungus ( Magnaporthe oryzae ) causes rice blast.

3. Application of (1) or (2) below in improving disease resistance of rice: (1) Used for specific gene knockout Ospis gRNA sequence, recombinant knockout vector or transformant; (2) Used for specific gene silencing Ospis Silencing fragments, recombinant silencing vectors or transformants; Gene Ospis The nucleotide sequence is shown in SEQ ID NO.1; The disease resistance is resistance to rice blast, which is caused by the blast fungus ( Magnaporthe oryzae ) causes rice blast.

4. A method for improving disease resistance of rice, characterized in that: include: Genes in rice Ospis knockout or silencing steps; Alternatively, the steps to delete the function of OsPIS, a negative regulatory protein of immunity in rice; Gene Ospis The nucleotide sequence is shown in SEQ ID NO.1; The amino acid sequence of the rice immune negative regulatory protein OsPIS is shown in SEQ ID NO. 2; The disease resistance is resistance to rice blast, which is caused by the blast fungus ( Magnaporthe oryzae ) causes rice blast.

5. The method according to claim 4, characterized in that Genes in rice are modified using CRISPR-Cas9 gene editing technology, VIGS technology, T-DNA insertion or RNA interference technology. Ospis Knockout or silencing.

6. Application of a mutant of the protein OsPIS in improving disease resistance in rice; characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.3 or SEQ ID NO.4; the disease resistance is resistance to rice blast, which is caused by rice blast fungus ( Magnaporthe oryzae ) causes rice blast.

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