Rice ribonuclease gene OsRNS4 and its encoded protein and application

Abstract

The present invention belongs to the field of plant genetic engineering, and specifically relates to a rice ribonuclease gene OsRNS4, a protein encoded therein, and an application thereof. The genomic nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.1, and the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.2. Experiments have shown that knocking out the rice ribonuclease gene OsRNS4 of the present invention reduces the disease resistance of rice to rice false smut, and the number of balls of rice false smut increases; overexpression can improve the disease resistance of rice to rice false smut, and the number of balls of rice false smut decreases, and transgenic plants with improved resistance to rice false smut can be obtained. Therefore, the ribonuclease gene and its protein in the present invention are of great significance for cultivating plants resistant to rice false smut, and are suitable for promotion and application.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and specifically relates to a rice ribonuclease gene OsRNS4 and a coded protein and application thereof. Background Art

[0002] Rice is an important food crop with a long history of cultivation. As a key research subject, rice disease resistance has always been a hot topic of concern, and the discovery and use of disease-resistant genes is the most economical and effective way to control rice diseases.

[0003] Ribonuclease (RNase) is a type of ribonucleolytic enzyme widely found in animals and plants. Its main physiological function is to control the type and quantity distribution of RNA in cells and participate in the post-transcriptional shearing, modification and degradation processes. At present, it has been found that RNase in some animals has anti-tumor, immunosuppressive, and angiogenesis functions. In addition, RNase is also related to the self-incompatibility, organogenesis and host defense mechanism of some plants. Among the plant RNase family that has been biochemically identified, only one RNase has other functions besides the general RNA degradation function. This type of RNase family, namely the T2 / S-RNase family, has been found in fungi, bacteria, slime molds, animals and plants, and is the most widely known type of RNase (Green., 1994, The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation, Plant J, doi:10.1046 / j.1365-313x.1994.6050673.x.).

[0004] In addition to S-RNases in the Solanaceae, Scrophulariaceae and Rosaceae families, plants also have other RNase T2 family ribonucleases, called S-like RNases, which have basically the same structure as S-RNases, but also have different conserved amino acid residues and perform different functions. S-like RNases have been found in all plant species, but except for the fact that they are not involved in self-incompatibility, their evolutionary process and other biological roles are still unknown.

[0005] There are eight members of S-like RNases in rice, namely OsRNS1-OsRNS8. Previous studies have found that when OsRNS4 is overexpressed in rice, the salt-alkali resistance of rice is improved (Zheng et al., 2014, Overexpression of an S-like ribonuclease gene, OsRNS4, confers enhanced tolerance to high salinity and hyposensitivity to phytochrome-mediated light signals in rice, Plant Sci, doi: 10.1016 / j.plantsci.2013.10.003.). However, it has not been found whether OsRNS4 is involved in the immune response process of rice, that is, the existing technology has not found the association between OsRNS4 and rice disease resistance. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a rice ribonuclease gene OsRNS4 and its encoded protein and application.

[0007] The first object of the present invention is to provide a rice ribonuclease gene OsRNS4, the genomic nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown as SEQ ID NO.1, the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown as SEQ ID NO.2, and the rice ribonuclease gene OsRNS4 is related to rice disease resistance.

[0008] The second object of the present invention is to provide a protein encoded by the rice ribonuclease gene OsRNS4, the amino acid sequence of the protein is shown in SEQ ID NO.3.

[0009] The third object of the present invention is to provide an expression vector of the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4.

[0010] The fourth object of the present invention is to provide a transgenic rice that overexpresses the rice ribonuclease gene OsRNS4.

[0011] The fifth object of the present invention is to provide a rice with a knockout gene of the rice ribonuclease gene OsRNS4.

[0012] Preferably, overexpression of the rice ribonuclease gene OsRNS4 can enhance resistance to rice false false disease; knocking out the rice ribonuclease gene OsRNS4 weakens the resistance of rice to rice false false disease.

[0013] A sixth object of the present invention is to provide a method for cultivating disease-resistant rice, comprising the following steps:

[0014] 1) transforming rice callus with the rice ribonuclease gene OsRNS4, wherein the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.2;

[0015] 2) Regenerating the transformed rice callus into rice;

[0016] 3) Cultivating regenerated rice and overexpressing the rice ribonuclease gene OsRNS4.

[0017] The seventh object of the present invention is to provide a method for detecting protein expression in transgenic plants of rice ribonuclease gene OsRNS4, comprising the following steps:

[0018] For the constructed rice ribonuclease gene OsRNS4 overexpression plants: extract the total protein from the leaves of the transgenic plants, and perform protein immunoblotting (Western Blot) on the extract. If the target band is detected, it indicates that the plant is transgenic positive, and if not, it indicates that the plant is transgenic negative.

[0019] The eighth object of the present invention is to provide a method for creating a rice OsRNS4 gene knockout material, comprising the following steps:

[0020] 1) designing primers according to the nucleotide sequence of the rice ribonuclease gene OsRNS4 and constructing an OsRNS4 gene knockout vector;

[0021] 2) Transform rice callus with OsRNS4 gene knockout vector;

[0022] 3) Regenerating the transformed rice callus into rice;

[0023] 4) Cultivating regenerated rice and knocking out the rice ribonuclease gene OsRNS4.

[0024] The ninth object of the present invention is to provide a method for detecting gene knockout of rice ribonuclease gene OsRNS4 transgenic plants, comprising the following steps:

[0025] For the constructed knockout plants: design primers according to the target, extract the genomic DNA of the leaves of the transgenic plants, use the primers to amplify the purified DNA fragments, and perform sequencing. If the sequencing results show a mutation and a single peak, it means that it is a transgenic positive homozygous plant.

[0026] The gene of the present invention is cloned from the genome nucleotide sequence of Oryza sativa L.ssp.japonicacv.Nipponbare. According to the analysis of Nipponbare in NCBI, the nucleotide sequence of OsRNS4 gene is shown in SEQ ID NO.1, the gene nucleotide is 1991bp in length, has 3 introns and 4 exons, its CDS segments are 62-175, 891-1052, 1136-1328 and 1442-1731 respectively, and the cDNA is 759bp in length.

[0027] Experiments have shown that knocking out the rice ribonuclease gene OsRNS4 of the present invention can reduce the resistance of rice to rice false smut, and increase the number of rice false smut balls; overexpression can improve the resistance of rice to rice false smut, and reduce the number of rice false smut balls, and transgenic plants with improved resistance to rice false smut can be obtained. Therefore, the ribonuclease protein and its gene in the present invention are of great significance for cultivating rice false smut-resistant plants and are suitable for promotion and application.

[0028] Beneficial effects of the present invention:

[0029] 1. The rice ribonuclease gene OsRNS4 is a good example of ribonuclease involvement in rice immunity, which has certain reference value for understanding the function of ribonucleases and the regulation of plant immunity.

[0030] 2. After OsRNS4 gene knockout, the resistance to rice false smut was weakened, and the number of rice false smut balls increased; after overexpression, the resistance to rice false smut was enhanced, and the number of rice false smut balls decreased; therefore, this gene can be used in breeding work.

[0031] 3. Since ribonucleases are a large family in plant cells, the study of the OsRNS4 gene has a good reference value for researchers to study other ribonucleases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The sequencing results of OsRNS4 gene knockout transgenic lines;

[0033] Figure 2 The results of protein expression detection of OsRNS4 gene overexpression transgenic strains;

[0034] Figure 3 Detection of OsRNS4 mRNA expression in overexpression lines

[0035] Figure 4 The results of the disease course-related gene expression test of the transgenic strains;

[0036] Figure 5The results of rice koji inoculation of transgenic lines; A is the appearance observation of rice ears, B is the statistical result of the incidence rate, and different letters indicate significant differences. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the technical means used are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used are all available from commercial channels.

[0038] Example 1: Construction of OsRNS4 gene knockout vector and overexpression vector and Agrobacterium-mediated genetic transformation

[0039] 1. Transgenic vector construction

[0040] 1.1 Construction of knockout vector

[0041] The nucleotides before the NGG sequence in the cDNA of the rice ribonuclease gene OsRNS4 were selected as the target to design primers. The sequences are as follows:

[0042] KO1-F: 5'-aaacCATGGTATGAACTGCGAATC-3', SEQ ID NO.4;

[0043] KO1-R: 5'-ggcaGATTCGCAGTTCATACCATG-3', SEQ ID NO.5;

[0044] KO2-F: 5'-aaacACAATCCTTTCGACATTAAC-3', SEQ ID NO.6;

[0045] KO2-R: 5'-ggcaGTTAATGTCGAAAGGATTGT-3, SEQ ID NO.7.

[0046] KO1-F and KO1-R are used as a pair of primers, and KO2-F and KO2-R are used as a pair of primers to construct knockout vectors. The specific construction methods are as follows:

[0047] The vectors used are pOs-sgRNA and pOs-cas9 (for both vectors, see Miao J et al., 2013, Targeted mutagenesis in rice using CRISPR-Cas system, Cell Res, doi:10.1038 / cr.2013.123). The primers were first diluted to 10 μM with ddH2O, and then the forward and reverse primers were mixed in equal volumes (25 μl each) and added to the PCR tube, with a final volume of 50 μl. Place it on the PCR instrument at 98°C for 2 min; 25°C for 10 min, allowing the primers to form double strands during slow cooling. pOs-sgRNA needs to be digested with NEB's Bsa I for 4 hours, and 0.5 μl Bsa I enzyme is added at 1.5 hours of digestion. The initial system refers to the 50 μl system in the instruction manual. Then, the gel was cut and recovered by electrophoresis. The connection system needs to consider the vector recovery concentration. If the recovery concentration is 30ng / μl, the electrophoresis recovery product of the PCR product of the primer self-ligation is about 1.5μl for every 15μl connection system. The remaining volume, except for the enzyme and buffer, is the pOs-sgRNA vector fragment. Connect overnight at 16℃. Transform with DH5α competent medium and screen with Kana (kanamycin). Then use LR reaction to recombinate the sequence into the pOs-cas9 vector. React at 25℃ overnight. After that, all reaction products are transformed into DH5α competent medium, and Spe (spectinomycin) is used to screen positive plasmids to obtain the OsRNS4 gene knockout vector.

[0048] The primer self-ligation system is as follows:

[0049] Forward primer 25 μl

[0050] Reverse primer 25 μl

[0051] On PCR instrument: 98℃ for 2 min; 25℃ for 10 min.

[0052] The vector enzyme digestion system is as follows:

[0053] pOS-sgRNA 2 μg

[0054] Bsa I 2μl (add 1.5μl first, add 0.5μl when the enzyme is digested for 1.5h)

[0055] Cutsmart Buffer 5μl

[0056] Add ddH2O to a total system of 50 μl and perform enzyme digestion at 37°C for 4 h.

[0057] The connection system is as follows:

[0058]

[0059] Ligation was performed overnight at 16°C.

[0060] The above method obtained two knockout vectors: the KO1 vector constructed by primers KO1-F and KO1-R, and the KO2 vector constructed by primers KO2-F and KO2-R.

[0061] 1.2 Construction of overexpression vector

[0062] The upstream primer and the downstream primer were designed by cutting a section of the nucleotides at both ends of the open reading frame of the rice ribonuclease gene OsRNS4. The sequences are as follows:

[0063] pC1305-3FLAG-RNS4-F:

[0064] 5'-ACA GAG CTC GGT ACC ATG GAG CAG AGG AAA TTT TTG TTG-3', SEQ IDNO.8;

[0065] pC1305-3FLAG-RNS4-R:

[0066] 5'-TTT GTA GTC AAG CTT GGC CAG CAC AGT TTC AGT GG-3', SEQ ID NO.9.

[0067] The vector used was pC1305 (Gangling Li et al., 2022, RGN1 controls grain number and shapes panicle architecture in rice, Plant Biotechnol J, doi: 10.1111 / pbi.13702), and the pC1305 vector was cut with KpnI / HindIII. The full length of the rice ribonuclease gene OsRNS4 open reading frame (SEQ ID No. 2 sequence) was amplified using the cDNA of Nipponbare as a template, and the vector after enzyme cutting and the sequence fragment of SEQ ID No. 2 were connected by homologous recombinase and transformed into Escherichia coli DH5α. After sequencing verification, the obtained vector is the OsRNS4 gene overexpression vector, and then the plasmid is extracted and heat-shocked into Agrobacterium EHA105. Pick a single clone for expansion culture, perform PCR verification, add an equal volume of 40% glycerol, mix well, and store at -80°C for later use. When constructing the vector, high-fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing Novozyme Biotechnology Co., Ltd.) was used for amplification to reduce the possibility of base mutation in the amplified sequence. PCR conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 60°C annealing and extension for 30 s / kb, 35 cycles; 16°C storage.

[0068] 2. Genetic transformation

[0069] The OsRNS4 gene knockout vector (KO1 vector and KO2 vector) and the OsRNS4 gene overexpression vector were introduced into the japonica rice variety Nipponbare by using the genetic transformation method mediated by Agrobacterium EHA105, including the transfer of the vector into the Agrobacterium strain EHA105, callus induction, subculture, pre-culture, Agrobacterium culture and suspension, infection and co-culture, Agrobacterium removal, callus screening, differentiation and transplanting, seedling hardening and transplanting, and detection. Referring to the method of "Hiei et al., 1994, Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6: 271-282", the specific steps are as follows:

[0070] 2.1 Screening of callus tissue

[0071] The dried Nipponbare callus was transferred to a solid selection medium (containing 400 mg / L hygromycin) for screening and cultured at 28°C for 30 days.

[0072] 2.2 Differentiation and transplantation

[0073] 1) The selected callus tissue was transferred to a differentiation medium containing hygromycin (50 mg / L) and cultured at 28°C for 30 days. 2) Green shoots with good growth were selected and transferred to a rooting medium.

[0074] 2.3 Hardening and transplanting

[0075] When the green seedlings grow to the three-leaf stage (about 15 days) and the root system is well developed, open the test tube and cover the surface of the culture medium with tap water to allow the seedlings to adapt to the external environment for 3 days. Wash off the solid culture medium at the roots of the seedlings, soak them in tap water for a week, and then the seedlings can be transplanted into the soil.

[0076] 2.4 Positive detection of transgenic plants

[0077] 1) Knockout transgenic plants can be confirmed as positive homozygous strains by sequencing in the T0 generation. Take the side leaves of the T0 seedlings, freeze them in liquid nitrogen and grind them. After extracting the genome, use the amplification primers of the gene to amplify and purify the genome fragments, and then send them for sequencing (Beijing Qingke Biotechnology Co., Ltd.). The results show a single peak and a mutation, which is a positive homozygous plant ( Figure 1 ).

[0078] 2) Take the lateral leaves of T0 seedlings of the overexpression strain, freeze them in liquid nitrogen and grind them, add an appropriate amount of 5×SDS loading buffer, and place them in a 100℃ metal bath for 10 minutes to release and denature the total protein. After completion, the samples are stored at -80℃ or directly run on denaturing gel. Gel electrophoresis is performed for about 2 hours, then transferred to the membrane and incubated with Anti-Flag antibody for verification ( Figure 2 ).

[0079] 2.5 Obtaining homozygous overexpression plants

[0080] For overexpressing plants, the recovered T1 generation seeds need to be screened on hygromycin 1 / 2MS plates to obtain a 3:1 trait separation. At this time, the wild type is eliminated, and the homozygous and heterozygous transgenic plant seedlings are planted again and the T2 generation seeds are harvested. They are screened again using hygromycin 1 / 2MS plates, and the strains without trait separation are homozygous.

[0081] Example 2: RNA level expression detection of overexpression strains

[0082] 1. RNA extraction (kit used is CWBIO, Kangwei Century Biotechnology Co., Ltd.):

[0083] 1) Take 2-3 leaves of transgenic rice plants cultured in 1 / 2MS for one week and transfer them to a clean 2mL centrifuge tube containing burned steel beads. 2) Quickly freeze in liquid nitrogen, then use a ball mill to completely crush the leaf samples into powder, add 1mL TRIZON, shake for 2min, and let the sample stand for 5min after fully mixing. 3) Add 200μL chloroform, shake vigorously for 15s, and let stand at room temperature for 2min. 4) Centrifuge at 12000rpm at 4℃ for 10min. 5) It can be seen that the solution in the centrifuge tube is divided into three layers. Take the supernatant to a new RNase-free centrifuge tube, add an equal volume of 70% ethanol prepared with RNase-free water, and mix by inversion. 6) Add to the adsorption column twice, centrifuge at 12000rpm at 4℃ for 15s, and discard the waste liquid. 7) Add 350μLRW1 to the adsorption column, centrifuge at 12000rpm at 4℃ for 15s, and discard the waste liquid. 8) Prepare the incubation solution (52μL RNase-Free Water + 8μL 10×Reaction Buffer + 20μL DNase I (1U / μL)) in advance. After the above centrifugation, add 80μL of incubation solution to the center of the adsorption column and let it stand at 28℃ for 15min. 9) After the incubation is completed, centrifuge at 12000rpm at 4℃ for 15s and discard the waste liquid. 10) Add 350μL RW1, centrifuge at 12000rpm at 4℃ for 15s and discard the waste liquid. 11) Add 500μL RW2, centrifuge at 12000rpm at 4℃ for 15s and discard the waste liquid. Repeat once after completion. 12) Centrifuge the empty column at 12000rpm at 4℃ for 2-3min. Transfer the column to a new RNase-free centrifuge tube and open the lid to dry for 3-5min. 13) Add 40 μL RNase-free water and centrifuge at 12000 rpm for 1 min to obtain RNA. After measuring the concentration, store it in a -80°C refrigerator.

[0084] 2. Reverse transcription:

[0085] The total reverse transcription system is 20 μL and is divided into two steps

[0086] RNA 2 μg

[0087] Oligo d(T)(100μM)1μL

[0088] RNA-free Water to 14μL

[0089] After mixing, put it into the PCR instrument and heat it at 72℃ for 10 minutes. After completion, quickly put it on ice for 3 minutes.

[0090] Add to the PCR tube in the previous step

[0091]

[0092] After gently mixing, put it into the PCR instrument, invert at 42℃ for 60min, and heat at 70℃ for 10min to stop the reaction. Take it out to obtain the cDNA solution, and use it immediately or store it in a -20℃ refrigerator.

[0093] 3. Fluorescence quantitative PCR:

[0094] The quantitative real time polymerase chain reaction (qRT-PCR) experimental method was based on FastSYBR Mixture, Cosmed Biotech Co., Ltd. The primers used were qRT-RNS4-F: 5'-ATGGAAGAGCGAGTGGAAC-3' (SEQ ID NO. 10) and qRT-RNS4-R: 5'-GTTGTACAGTTGGTAGTCCGG-3' (SEQ ID NO. 11).

[0095] 1) PCR reaction system

[0096] Reagents (20 μL reaction system)

[0097]

[0098]

[0099] 2) PCR reaction conditions

[0100] Pre-denaturation 95℃30s

[0101] Denaturation 95℃5s

[0102] Annealing / extension 60℃30s

[0103] The denaturation and annealing / extension steps were repeated for 40 cycles.

[0104] The quantitative results were analyzed using Bio-Rad CFX Manager. First, the dissolution curve, QC (quality control), etc. were analyzed, and substandard data were removed. The Gene Study function of the software was used to press (1+E) -ΔΔ Ct algorithm to analyze the expression of target genes ( Figure 3 ). Figure 3 The results showed that after transformation, the OsRNS4 gene overexpression vector we constructed had a higher expression level than the wild type.

[0105] Example 3: PR gene detection in transgenic lines

[0106] The expression of pathogenesis-related genes is a typical experiment to detect the basic immune phenotype of plants. Therefore, we tested the PR genes as follows:

[0107] 1. Rice seed disinfection

[0108] Put the seeds into a thresher to peel them. Move the peeled seeds to a sterilized 150mL Erlenmeyer flask in a clean bench, add 70% ethanol, and shake gently for 3 minutes. Pour out the ethanol solution, add 50% sodium hypochlorite, 28℃+120rpm, 30 minutes (this step is done on a shaker). Pour out the sodium hypochlorite solution, wash with sterile distilled water at least 12 times, 30 seconds each time. Shake the flask gently during the process, and try not to let the seeds flow out when pouring out the waste liquid. Spread the washed seeds on sterilized filter paper, adjust the clean bench wind slightly, and blow the seeds dry, but not for too long to prevent seed contamination. Use directly or collect in a clean 50mL tube and store in a 4℃ refrigerator.

[0109] 2. Elicitor treatment

[0110] The sterilized seeds were spread on 1 / 2MS solid medium and cultured for 3 days, then the seedlings with the same status were selected and transferred to 1 / 2MS liquid medium for further culture for 3 days. The rice seedlings were treated with the elicitor Chitin for 6 hours, and then the same side leaf was taken, and at least three leaves were taken from each strain for the next experiment.

[0111] 3. RNA extraction, reverse transcription and fluorescence quantitative PCR detection (specific steps are the same as Example 2). The PR gene primers are PR5-qF: 5'-CAACAGCAACTACCAAGTCGTCTT-3' (SEQ ID NO.12), PR5-qR: 5'-CAAGGTGTCGTTTTATTCATCAAC-3' (SEQ ID NO.13). The results showed that the PR gene level of the knockout strain induced by the elicitor was significantly lower than that of the wild type, while the PR gene level of the overexpression strain was significantly higher than that of the wild type ( Figure 4 ).

[0112] Example 4: Transgenic lines inoculated with rice koji

[0113] Take out the rice smut pathogen JS60-2 (Zheng et al., 2022, Ustilaginoideavirens secretes a family of phosphatases that stabilize the negative immune regulator OsMPK6 and suppress plant immunity, Plant Cell, doi:10.1093 / plcell / koac154.) from the -80℃ ultra-low temperature freezer and activate it on the PSA plate, and culture it in a 28℃ incubator for 10 days. Cut the bacterial block into 100mL PS culture medium and culture it in a shaker at 28℃ at 180rpm for 5 days. Break the mycelium in the rice smut pathogen solution to form a spore mycelium mixture. Then adjust the spore concentration to 1×10 with sterile PS liquid. 6 / mL. Use a syringe to inject 1mL of bacterial solution into the rice ears of KO-1 (KO1 knockout vector transformant), KO-2 (KO2 knockout vector transformant), OE-1 (overexpression vector transformant) and WT (Nipponbare plant, wild type) (select rice ears 5 days before heading), and inoculate 10 replicates for each sample. Count the number and incidence of rice balls 21 days after inoculation.

[0114] The results showed that compared with the wild type, the knockout strain had significantly reduced resistance to rice false smut, while the overexpression strain had significantly increased resistance to rice false smut ( Figure 5 ), indicating that the OsRNS4 gene can regulate rice resistance to rice false smut.

[0115] It should be noted that:

[0116] The sequence of SEQ ID NO.1 is as follows:

[0117]

[0118]

[0119] The sequence of SEQ ID NO.2 is as follows:

[0120]

[0121] The sequence of SEQ ID NO.3 is as follows:

[0122]

[0123] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the attached claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0124] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of rice ribonuclease gene OsRNS4 in rice germplasm resource improvement, characterized in that: The application is for enhancing the resistance of rice to rice false false disease. Overexpression of the rice ribonuclease gene OsRNS4 can enhance the resistance to rice false false disease. The nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.1, and the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.

2.

2. The use of the rice ribonuclease gene OsRNS4 in rice germplasm resource improvement according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.

3.

3. The use of the rice ribonuclease gene OsRNS4 in rice germplasm resource improvement according to claim 1, characterized in that: The method for cultivating rice with overexpression of OsRNS4 to enhance the resistance of rice to rice false smut disease comprises the following steps: 1) The rice ribonuclease gene OsRNS4 is transferred into rice callus, wherein the nucleotide sequence of the open reading frame of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.2; 2) Regenerating the transformed rice callus into rice; 3) Cultivating regenerated rice and overexpressing the rice ribonuclease gene OsRNS4.

4. The use of the rice ribonuclease gene OsRNS4 in rice germplasm resource improvement according to claim 3, characterized in that: Cultivating the regenerated rice and over-expressing the rice ribonuclease gene OsRNS4 to obtain a transgenic plant over-expressing the rice ribonuclease gene OsRNS4; A method for detecting protein expression in transgenic plants overexpressing the rice ribonuclease gene OsRNS4 comprises the following steps: The total protein of the leaves of the transgenic plants overexpressing the rice ribonuclease gene OsRNS4 was extracted, and the extract was subjected to protein immunoblotting. If the target band was detected, it indicated that the plant was transgenic positive, and if not, it indicated that the plant was transgenic negative.

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