Application of rice gene OsZP2 in disease resistance

Abstract

The invention discloses application of a rice gene OsZP2 in regulation and control of rice disease resistance, the gene is used for positively regulating and controlling rice sheath blight resistance and rice blast resistance, specifically, the OsZP2 gene from japonica rice variety Nipponbare (NIP) is introduced into indica rice variety by utilizing a transgenosis method, and rhizoctonia solani and rice blast are inoculated respectively, so that the rice disease resistance of rice is regulated and controlled. The disease spot area or length of the transgenic (OsZP2) plant is reduced, and the disease resistance is enhanced; the OsZP2 gene of NIP is introduced into an indica rice variety by molecular marker-assisted selection to obtain a single-fragment introgression line containing the OsZP2 gene, rhizoctonia solani and magnaporthe oryzae are inoculated respectively, the area or length of disease spots of a plant containing the introgression line containing the OsZP2 gene is reduced, and the disease resistance is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of rice gene OsZP2 in disease resistance. Background Art

[0002] Rice (Oryza sativa L.) is one of the most important crops in the world, feeding more than half of the world's population. Sheath blight caused by the strong saprophytic fungus Rhizoctonia solani Kühn is one of the most important diseases of rice, mainly damaging the leaf sheaths of rice, and the leaves can also be diseased, resulting in the death of the leaves due to water loss, and ultimately causing a significant reduction in yield and quality. In the 1970s of the last century, sheath blight was listed as a national prevention and control target in China, and together with rice blast and bacterial blight, they are collectively referred to as the three major diseases of rice. Traditional chemical control methods are costly, highly polluting, harmful to the environment and human body, and at the same time promote the evolution and drug resistance of pests and pathogens, posing a greater threat to rice production and even posing a potential hazard to human safety. Therefore, exploring rice disease-resistant genes and cultivating rice varieties with disease resistance is the most effective measure to control pests and diseases and improve rice production, and has important scientific significance and application value.

[0003] Previous studies have shown that the resistance of rice to sheath blight is mostly a typical quantitative trait, controlled by multiple genes or quantitative trait loci (QTL). More than 60 QTLs for sheath blight resistance have been identified and reported, and these QTLs are distributed unevenly on 12 chromosomes of rice. However, since sheath blight resistance is also significantly affected by agronomic traits such as growth period and plant height, the authenticity of most QTLs remains to be further verified. With the development of molecular biology and functional genomics, researchers in rice have successively elucidated a large number of candidate genes related to sheath blight resistance through technical means such as gene overexpression, RNAi, T-DNA insertion, gene editing, and comparative transcriptomics, and functionally identified some genes by transgenic methods. OsNAC4 is a plant-specific transcription factor, and it has been confirmed that it negatively regulates the resistance of rice to sheath blight; OsXNDL2 positively regulates the sheath blight resistance of rice by activating the ethylene signaling pathway; Tiwari et al. identified a tau-class GST gene OsGSTU5 that can positively regulate the sheath blight resistance of rice; Lin et al. reported that OsPP2A-1 strengthens the resistance of rice to sheath blight by positively regulating the expression of defense genes such as OsPBZ1 and OsPR1b; through transgenic analysis of the chlorophyll metabolism gene OsNYC3, it was found that OsNYC3 negatively regulates the sheath blight resistance of rice, and at the same time, inhibiting the expression of this gene basically does not affect the main agronomic traits, suggesting its important application prospects in the molecular breeding of rice for sheath blight resistance.

[0004] The present invention isolated a gene OsZP2, which encodes a papain of the cysteine protein family, has an amino acid similarity of up to 89.3% with the ginger protein zingipain-2, is an allele derived from japonica rice, and has the effect of increasing the disease resistance of rice. By using the transgenic method, the OsZP2 gene derived from japonica rice NIP was complemented and expressed in the ZS97 variety, and it was found that the complemented ZS97 had the ability to resist sheath blight and rice blast. By using the method of molecular marker-assisted selection, the OsZP2 gene derived from japonica rice variety NIP was introduced into rice variety ZS97, and it could also increase its ability to resist sheath blight and rice blast. Summary of the Invention

[0005] The object of the present invention is to provide a new use of the rice gene OsZP2. Specifically, it is the application of the gene OsZP2 in positively regulating the ability of rice to resist sheath blight or / and rice blast.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of the OsZP2 gene in improving the disease resistance of rice, and the full-length sequence of the OsZP2 gene is shown in SEQ ID NO.1. Specifically, the disease resistance includes the ability to resist sheath blight and rice blast.

[0008] A method for improving the ability of rice to resist sheath blight or / and rice blast, which is to introduce the OsZP2 gene into indica rice varieties by transgenic methods, construct a complementary vector of the OsZP2 gene, and introduce the OsZP2 gene into indica rice varieties by Agrobacterium-mediated transformation; or, using japonica rice varieties as donor parents and indica rice varieties as recipient parents, crossing the recipient parents with the donor parents, and then continuously backcrossing the hybrid offspring with the recipient parents for more than 3 generations, combined with molecular marker-assisted screening to screen single segment introgression line plants containing the OsZP2 gene.

[0009] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0010] 1. The present invention clarifies the excellent parent of the disease resistance gene OsZP2. The OsZP2 allele derived from japonica rice NIP has the effect of enhancing the sheath blight resistance of rice; by using the transgenic method, the OsZP2 gene derived from japonica rice NIP is complementarily expressed in ZS97, and it also has the effect of enhancing the sheath blight resistance. By using molecular marker-assisted selection and gene chip technology to screen single segment introgression lines containing the OsZP2 gene, it also has the effect of enhancing the sheath blight resistance.

[0011] 2. The single segment introgression line of OsZP2 obtained in the present invention can be directly used as a donor in rice disease resistance breeding.

[0012] 3. The present invention clarifies that the disease-resistant gene OsZP2 also has the ability to resist other fungal diseases, such as Magnaporthe oryzae. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram for the construction of the OsZP2 recombinant vector. The gene OsZP2 is ligated to the complementary vector PC1301 to form a complementary recombinant vector, and this gene consists of 3985 bases.

[0014] Figure 2 shows the positive detection results of T0 and T1 complementary transgenic plants. The results of PCR detection of T0 and T1 individual plants using the specific primers GUS1.2F and GUS1.2R of the GUS reporter gene on the complementary vector PC1301, Figure 2.1 are the detection results of the T0 generation, Figure 2.2 are the detection results of the T1 generation.

[0015] Figure 3 It is the construction process of the single fragment introgression line of the OsZP2 gene.

[0016] Figure 4 It is the genotype comparison of the whole genomes of the recipient parent ZS97 and the single fragment introgression line plant ZS97-NIL-OsZP2 containing the OsZP2 gene.

[0017] Figure 5 It is the comparison of the lesion areas of the leaf sheaths inoculated with Rhizoctonia solani of two complementary family positive lines (numbered OsZP2-CP1+ and OsZP2-CP2+ respectively) and their corresponding negative control lines (OsZP2-CP1- and OsZP2-CP2-). ** indicates that the t-test between the complementary positive and its corresponding negative line reaches a significant level of 0.01.

[0018] Figure 6 It is the comparison of the lesion areas of the leaf sheaths inoculated with Rhizoctonia solani of the recipient parent ZS97 and the single fragment introgression line plant ZS97-NIL-OsZP2 containing the OsZP2 gene. ** indicates that the t-test between the introgression line and its corresponding negative recipient parent ZS97 line reaches a significant level of 0.01.

[0019] Figure 7 It is the comparison of the lesion lengths of the leaves inoculated with Magnaporthe oryzae of two complementary family positive lines (numbered OsZP2-CP1+ and OsZP2-CP2+ respectively) and their corresponding negative control lines (OsZP2-CP1- and OsZP2-CP2-). ** indicates that the t-test between the complementary positive and its corresponding negative line reaches a significant level of 0.01.

[0020] Figure 8Comparison of the lesion lengths on the leaves of the recipient parent ZS97 and the single segment introgression line plant ZS97-NIL-OsZP2 containing the OsZP2 gene after inoculation with Magnaporthe oryzae. ** indicates that the t-test between the introgression line and its corresponding negative recipient parent ZS97 line reaches a significant level of 0.01. Detailed implementation manners

[0021] The present invention is given by the following detailed examples. Based on the following description and these examples, those skilled in the art can determine the basic features of the present invention, and various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention so as to make it applicable to various uses and conditions. The molecular cloning methods and reagent formulations are all referred to "Molecular Cloning: A Laboratory Manual" (J. Sambrook et al., Third Edition, Science Press, 2002) unless otherwise specified; primer synthesis and sequencing work were completed by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd.

[0022] The culture media for genetic transformation used in the present invention and the methods for preparing them are described as follows:

[0023] (1) Abbreviations of reagents and solutions

[0024] The abbreviations of the phytohormones used in the culture media in the present invention are as follows:

[0025] 6-BA (6-BenzylaminoPurine, 6-benzyladenine)

[0026] CN (Carbenicillin, carbenicillin)

[0027] KT (Kinetin, kinetin)

[0028] NAA (Napthalene acetic acid, naphthaleneacetic acid)

[0029] IAA (Indole-3-acetic acid, indole-3-acetic acid)

[0030] 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid)

[0031] AS (Acetosringone, acetosyringone)

[0032] CH (Casein Enzymatic Hydrolysate, casein enzymatic hydrolysate)

[0033] HN (Hygromycin B, hygromycin B)

[0034] DMSO (Dimethyl Sulfoxide, Dimethyl Sulfoxide)

[0035] MSmax (MS Macronutrient Component Solution)

[0036] MSmix (MS Micronutrient Component Solution)

[0037] (2) Main Solution Formulations

[0038] 1) Subculture A Mother Liquid Stock Solution (Prepared as 100× Concentrate)

[0039]

[0040] Add distilled water to make up to 1000 ml and store at room temperature for later use.

[0041] 2) Subculture B Mother Liquid Stock Solution (Prepared as 100× Concentrate)

[0042]

[0043]

[0044] Add distilled water to make up to 1000 ml and store at room temperature for later use.

[0045] 3) Iron Salt (Fe2EDTA) Stock Solution (Prepared as 100× Concentrate)

[0046] Dissolve 3.73 g of disodium ethylenediaminetetraacetate (Na2EDTA·2H2O) and 2.78 g of FeSO4·7H2O separately, mix and make up to 1000 ml with distilled water, warm bath at 70°C for 2 hours, and store at 4°C for later use.

[0047] 4) Vitamin Stock Solution (Prepared as 100× Concentrate)

[0048]

[0049] Add distilled water to make up to 1000 ml and store at 4°C for later use.

[0050] 5) MS Medium Macronutrient Mother Liquid (MSmax Mother Liquid) (Prepared as 10× Concentrate)

[0051]

[0052] Dissolve the above reagents at room temperature and make up to 1000 ml with distilled water.

[0053] 6) MS Medium Micronutrient Mother Liquid (MSmin Mother Liquid) (Prepared as 100× Concentrate)

[0054]

[0055] Dissolve the above reagents at room temperature and make up the volume to 1000 ml with distilled water.

[0056] 7) Preparation of 2,4-D stock solution (1 mg / ml)

[0057] Weigh 100 mg of 2,4-D, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water, dissolve completely and make up the volume to 100 ml, and store at room temperature.

[0058] 8) Preparation of 6-BA stock solution (1 mg / ml)

[0059] Weigh 100 mg of 6-BA, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water, dissolve completely and make up the volume to 100 ml, and store at room temperature.

[0060] 9) Preparation of naphthaleneacetic acid (NAA) stock solution (1 mg / ml)

[0061] Weigh 100 mg of NAA, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water, dissolve completely and make up the volume to 100 ml, and store at 4 °C for later use.

[0062] 10) Preparation of indoleacetic acid (IAA) stock solution (1 mg / ml)

[0063] Weigh 100 mg of IAA, dissolve it in 1 ml of 1N potassium hydroxide for 5 minutes, then add 10 ml of distilled water, dissolve completely and make up the volume to 100 ml, and store at 4 °C for later use.

[0064] 11) Preparation of glucose stock solution (0.5 g / ml)

[0065] Weigh 125 g of glucose, then dissolve it with distilled water and make up the volume to 250 ml, sterilize and store at 4 °C for later use.

[0066] 12) Preparation of AS stock solution

[0067] Weigh 0.392 g of AS, add 10 ml of DMSO to dissolve, dispense into 1.5 ml centrifuge tubes, and store at 4 °C for later use.

[0068] 13) 1N potassium hydroxide stock solution

[0069] Weigh 5.6 g of potassium hydroxide, dissolve it with distilled water and make up the volume to 100 ml, and store at room temperature for later use.

[0070] 14) AA macroelement mother liquor (AAmax mother liquor) (prepared according to 10× concentrated solution)

[0071]

[0072] Make up the volume to 1000 ml with distilled water and store in the dark at room temperature.

[0073] 15) AA Trace Element Mother Solution (AAmin Mother Solution) (prepared according to 100× concentrated solution)

[0074]

[0075]

[0076] Make up the volume to 1000 ml with distilled water and store in the dark at room temperature.

[0077] (3) Medium Formulas for Rice Genetic Transformation

[0078] 1) Induction Medium

[0079]

[0080] Add 600 - 700 ml of H2O and adjust the pH to 6.0 with potassium hydroxide. After boiling, make up the volume to 1000 ml with H2O. Aliquot into 50 - ml Erlenmeyer flasks (25 ml / flask), seal with sealing film and sterilize.

[0081] 2) Subculture Medium

[0082]

[0083]

[0084] Add 900 ml of H2O and adjust the pH to 6.0 with potassium hydroxide. After boiling, make up the volume to 1000 ml with water. Aliquot into 50 - ml Erlenmeyer flasks (25 ml / flask), seal with sealing film and sterilize.

[0085] 3) Pre - culture Medium

[0086]

[0087] Add 250 ml of H2O and adjust the pH to 5.6 with potassium hydroxide. Seal with sealing film and sterilize.

[0088] Before use, dissolve the medium by boiling, add 5 ml of glucose stock solution (sterilized 50% glucose solution) and 250 μl of AS stock solution, then aliquot into petri dishes (25 ml / dish).

[0089] 4) Co - culture Medium

[0090]

[0091] Add 250 ml of H2O and adjust the pH to 5.6. Seal with parafilm and sterilize.

[0092] Before use, dissolve the medium by boiling, add 5 ml of glucose stock solution (sterilized 50% glucose solution) and 250 μl of AS stock solution, and then dispense into petri dishes (25 ml / dish).

[0093] 5) Suspension medium

[0094]

[0095] Add 100 ml of H2O and adjust the pH to 5.4. Filter sterilize, add 100 μl of AS stock solution, and dispense into two 100-ml Erlenmeyer flasks (50 ml / flask).

[0096] 6) Selection medium

[0097]

[0098] Add 250 ml of H2O and adjust the pH to 6.0. Seal with parafilm and sterilize.

[0099] Before use, dissolve the medium by boiling, add 250 μl of Hn and 200 ppm of CN, and then dispense into petri dishes (25 ml / dish).

[0100] 7) Differentiation medium

[0101]

[0102]

[0103] Add 1000 ml of H2O and adjust the pH to 6.0 with sodium hydroxide. After boiling, dispense into 100-ml Erlenmeyer flasks (50 ml / flask), seal with parafilm and sterilize.

[0104] 8) Rooting medium

[0105]

[0106] Add distilled water to 900 ml and adjust the pH value to 5.8 with 1N potassium hydroxide.

[0107] Boil and make up to 1000 ml with distilled water. Dispense into rooting tubes (25 ml / tube), seal, and sterilize by the above method.

[0108] 9) PDA medium

[0109] Potato 200 g / L, glucose 20 g / L, agar powder 15 g / L. Add 1000 ml of H2O, dispense into 250-ml Erlenmeyer flasks (150 ml / flask), seal, and sterilize.

[0110] Example 1: Isolation and cloning of the OsZP2 gene

[0111] Extract the DNA of the japonica rice variety NIP, and perform polymerase chain reaction (PCR) using the primers OsZP2-F1 (5'-GATTACGAATTCGAGCTCGGTACCATACTCCCCTATTGACTTTT-3') and OsZP2-R1 (5'-TGCAGGTCGACTCTAGAGGATCCCATTTTCCTTGTTGTTGCTT-3'). PCR program: pre-denature at 94°C for 5 minutes, 35 cycles (denature at 94°C for 40 seconds, anneal at 58°C for 40 seconds, extend at 72°C for 4.5 minutes), extend at 72°C for 10 minutes; sequence the PCR amplification product to obtain the full-length sequence of the OsZP2 gene of the NIP variety, including the sequence region 2000 kb upstream of the start codon and 500 bp downstream of the stop codon, and its sequence is shown in SEQ ID NO.1.

[0112] Example 2: Construction of the OsZP2 recombinant vector and establishment of Agrobacterium transformation

[0113] Double digest the PCR amplification product of OsZP2-F1 / R1 and the PC1301 empty vector with KpnⅠ and BamHⅠ, respectively recover the OsZP2 gene fragment and the PC1301 vector, and ligate them with T4 ligase to form a recombinant vector ( Figure 1 ). The above restriction endonucleases and T4 ligase are all purchased from Takara.

[0114] Transform the recombinant vector into Escherichia coli competent DH5ɑ (product of Takara), pick monoclonal colonies to extract plasmids, sequence with the OsZP2-F1 / R1 primers, and pick the correct plasmid to transform Agrobacterium EHA105 (product of Takara). The transformed Agrobacterium strain is named PC1301-OsZP2.

[0115] Example 3: Agrobacterium-mediated genetic transformation

[0116] (1) Callus induction

[0117] Remove the hulls of the mature seeds of the rice variety Zhenshan 97 (ZS97), and then treat them with 70% (v / v) ethanol for 1 minute and surface sterilize them with 0.15% (w / v) mercuric chloride (HgCl2) for 15 minutes in sequence; wash the seeds with sterile water 4-5 times; place the seeds on the indica rice induction medium; place the inoculated medium in the dark for 4 weeks at a temperature of 25 ± 1°C.

[0118] (2) Subculture

[0119] Select bright yellow, firm and relatively dry embryogenic calli, place them on the indica rice subculture medium and culture in the dark for 2 - 3 weeks at a temperature of 25 ± 1°C.

[0120] (3) Pre - culture

[0121] Select firm and relatively dry embryogenic calli, place them on the indica rice pre - culture medium and culture in the dark for 3 - 4 days at a temperature of 25 ± 1°C.

[0122] (4) Agrobacterium culture

[0123] Pre - culture the Agrobacterium strain PC1301 - OsZP2 on the LA medium with kanamycin resistance (product of Shanghai Sangon Biotech Co., Ltd.) for two days at a temperature of 28°C; scrape the Agrobacterium into the suspension medium and culture it in suspension at a temperature of 28°C.

[0124] (5) Infection

[0125] Transfer the pre - cultured calli into a sterilized bottle; adjust the suspension of Agrobacterium PC1301 - OsZP2 to OD 600 0.8 - 1.0; immerse the calli in the Agrobacterium suspension for 30 minutes; transfer the calli to sterilized filter paper to absorb the liquid; then place them on the indica rice co - culture medium and culture for 3 days at a temperature of 19 - 20°C.

[0126] (6) Screening

[0127] Wash the calli 8 times with sterilized water; immerse them in sterilized water containing 400 mg / L carbenicillin (CN) (product of Shanghai Sangon Biotech Co., Ltd.) for 30 minutes; transfer the calli to sterilized filter paper to absorb the liquid; transfer the calli to the indica rice selection medium containing 250 mg / L carbenicillin (CN) and 50 mg / L hygromycin (Hn) (product of Roche) and select and culture them 2 - 3 times, 2 weeks each time.

[0128] (7) Differentiation

[0129] Transfer the resistant calli to the indica rice differentiation medium and culture them under light at a temperature of 26°C.

[0130] (8) Rooting

[0131] Cut off the roots generated during the differentiation of the regenerated seedlings; then transfer them to the rooting medium and culture them under light for 2 - 3 weeks at a temperature of 26°C.

[0132] (9) Transplanting

[0133] Wash off the residual medium on the roots of the regenerated plants, transplant them into pots for potting, and keep the moisture moist in the first few days. Then transfer them to the field after the plants survive and grow strong.

[0134] Example 4: Identification of OsZP2 gene - transformed complementary plants

[0135] A total of 15 T0 generation OsZP2 transgenic complementary plants obtained from Example 3 were planted in the field. The DNA of the T0 generation plants was extracted, and the specific primers GUS1.2F (primer sequence: 5'-ACGACTCGTCCGTCCTGTAGAA-3') and GUS1.2R (primer sequence: 5'-CGGTTCGTTGGCAATACTCC-3') of the GUS reporter gene on the PC1301 vector were used for PCR detection of transgenic positive plants. The PCR program was pre-denaturation at 94°C for 5 minutes, 30 cycles (denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 1 minute), extension at 72°C for 7 minutes. The PCR products were detected by 1% agarose gel electrophoresis. The single plants that could amplify a 1200bp band were complementary positive single plants ( Figure 2.1 ). The complementary positive single plants showed segregation in the T1 generation. After the seeds of the offspring of the same plant line were sown, the GUS1.2 primers were used for amplification again. Similarly, the single plants that could amplify a 1200bp band were complementary positive single plants, and the single plants that could not amplify a 1200bp band were negative plant lines corresponding to the complementary positive plant lines ( Figure 2.2 ).

[0136] Example 5: Creation and identification of single fragment introgression lines of OsZP2 gene

[0137] Using ZS97 (Zhenshan 97) as the recipient parent and the japonica rice variety NIP as the donor parent, after one hybridization, and then continuously backcrossing with the recipient parent ZS97 for 3 generations, combined with molecular marker (KASP marker) and gene chip (GSR40K) selection. The three groups of molecular marker primers are shown in SEQ ID NO.2-4, SEQ ID NO.5-7, and SEQ ID NO.8-10 respectively. The molecular markers are used to detect whether the introgressed fragments in the populations of each hybridization generation are consistent with the donor parent, and the gene chip is used to screen for target fragments and eliminate background fragments. Finally, a single fragment introgression line with the genotype of the OsZP2 gene segment replaced by the genotype of NIP and the genetic background of other genomes being ZS97 was obtained, named ZS97-NIL-OsZP2, and the construction process is as Figure 3 shown. The whole genome genotypes of the recipient parent ZS97 and the single fragment introgression line plant ZS97-NIL-OsZP2 containing the OsZP2 gene were compared using a gene chip, and the results are shown in Figure 4.

[0138] Example 6: OsZP2 gene is involved in resistance to sheath blight of rice

[0139] The seeds of the transgenic complementary families OsZP2-CP1 and OsZP2-CP2 obtained in Example 4, the single segment introduction line ZS97-NIL-OsZP2 obtained in Example 5, and the wild type ZS97 were sown until the end of tillering, and the stems were taken for the sheath blight inoculation experiment, and the lesion area after inoculation was counted. The specific method is as follows:

[0140] (1) Preparation of in vitro stems: Cultivate rice in the field under natural temperature and light conditions until the booting stage, cut the main stems of healthy plants, trim the stems, and only retain the leaf sheaths and stem nodes of the first and second leaves. Cut 10 stems for each identification material in this way and place them in clean water for 12 hours; then vertically insert the stems into a round pot filled with nutrient soil at a certain interval, and place the round pot in a white bread basin filled with clean water, keeping the water layer in the basin to cover half of the round pot;

[0141] (2) Cultivation of sheath blight pathogen: Inoculate a mycelium block of sheath blight pathogen (Rhizoctonia solani Kühn) WH-1 into a PDA medium and culture it in the dark at 28°C for 2-3 days. Transfer one activated mycelium block to a new PDA medium for subculture and culture it in the dark at 28°C for 2 days. Take it out after the surface of the medium is covered with white mycelium.

[0142] (3) Inoculation of sheath blight: Take a mycelium block with a diameter of 5 mm and uniform thickness, gently spread the leaf sheath of the second leaf with your hands, take the mycelium block with tweezers, and put it into the spread leaf sheath, then carefully restore the leaf sheath to its original state, wrap the leaf sheath with moist cotton, and fix it with plastic wrap;

[0143] (4) Temperature and humidity management after inoculation: The white bread pot was placed in the culture room of the breeding factory during the whole growth period of the plant. The room temperature of the breeding factory was adjusted to 30°C / 26°C, the lighting conditions were 14h light / 10h dark, and a humidifier was installed in the culture room to ensure that the internal humidity was maintained at 75% to 95%;

[0144] (5) Investigation and recording of lesions: Pay close attention to the growth progress of lesions. Seven days after inoculation, measure and record the lesion area of ​​each inoculated stem.

[0145] After inoculation with the sheath blight pathogen WH-1, the lesion area of ​​the complementary positive strains (OsZP2-CP1+ and OsZP2-CP2+) was significantly lower than that of the corresponding negative control strains (OsZP2-CP1- and OsZP2-CP2-). Similarly, the lesion area of ​​the introduced material ZS97-NIL-OsZP2 was significantly reduced compared with the wild-type ZS97 ( Figure 5 and 6 ). This indicates that the OsZP2 gene is involved in rice resistance to sheath blight.

[0146] Example 7: The OsZP2 gene is involved in rice blast resistance

[0147] Seeds of the transgenic complementation lines OsZP2-CP1 and OsZP2-CP2 obtained in Example 4, the single segment introgression line NIL-OsZP2 obtained in Example 5, and the wild type ZS97 were sown. At the late tillering stage, rice leaves were taken, the leaf tips were removed, and the leaves were cut into 5-cm-long segments. Three small holes were pricked on the front of each small leaf with a 10-μl pipette tip to pierce the surface cells. The leaves were then placed in a petri dish containing 30 ml of 6BA solution (1 μg / ml), and 5 μl of the spore suspension of Magnaporthe oryzae I10-2 (8×10 5 ~1×10 6 spores / ml) was dropped onto each puncture site. The petri dish was covered, shaded with black plastic film for 24 hours, and cultured at about 25°C. The lesion length was measured after 5 days.

[0148] After inoculation with Magnaporthe oryzae I10-2, the lesion areas of the complementary positive lines (OsZP2-CP1+ and OsZP2-CP2+) were significantly lower than those of their corresponding negative control lines (OsZP2-CP1- and OsZP2-CP2-). Similarly, compared with the wild type ZS97, the lesion length of the introgression line ZS97-NIL-OsZP2 was significantly reduced ( Figure 7 and 8 ). This indicates that the OsZP2 gene is involved in the rice blast resistance function.

Claims

1. OsZP2 Use of a gene in enhancing the disease resistance of rice, characterized in that, The said OsZP2 The full-length gene sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein The disease resistance ability described is the ability to resist sheath blight or / and rice blast.

3. A method for improving the sheath blight resistance and / or blast resistance of rice, characterized in that, Introduce the gene described in claim 1 OsZP2 into indica rice varieties, and the resistance of the indica rice varieties to sheath blight and rice blast is enhanced.

4. The method according to claim 3, wherein Construct OsZP2 a complementary vector of the gene and introduce the OsZP2 gene into indica rice varieties by Agrobacterium-mediated transformation.

5. The method according to claim 3, wherein Using a japonica rice variety as the donor parent and an indica rice variety as the recipient parent, crossing the recipient parent with the donor parent, and then continuously backcrossing the hybrid offspring with the recipient parent for more than 3 generations. Combining molecular marker-assisted screening, introduce OsZP2 gene under the background of the recipient parent to obtain OsZP2 single segment introgression line plants of the gene.

6. The method according to claim 5, characterized in that, It includes three molecular markers, and the primer pairs of the molecular markers are shown as SEQ ID NO.2-4, SEQ ID NO.5-7, and SEQ ID NO.8-10.

7. Use of the single segment introgression line indica rice containing the gene recited in claim 1 in rice disease resistance breeding. OsZP2 ​

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