CN116083460A

CN116083460A - Rice ribonuclease gene OsRNS4 and encoding protein and application thereof

Info

Publication number: CN116083460A
Application number: CN202310063688.7A
Authority: CN
Inventors: 孙文献; 高涵; 汪激扬; 崔福浩; 方安菲; 郑馨航; 邱姗姗
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-02-06
Filing date: 2023-02-06
Publication date: 2023-05-09

Abstract

The invention belongs to the field of plant genetic engineering, and in particular relates to a rice ribonuclease gene OsRNS4 and a coding protein and application thereof. The genome nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown as SEQ ID NO.1, and the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown as SEQ ID NO. 2. Experiments prove that the disease resistance of rice to false smut can be reduced after the rice ribonuclease gene OsRNS4 is knocked out, and the balling number of the false smut is increased; after the over-expression, the disease resistance of the rice to the false smut can be improved, the balling number of the false smut is reduced, and a transgenic plant with improved resistance to the false smut can be obtained. Therefore, the ribonuclease gene and the ribonuclease protein have important significance for cultivating the false smut-resistant plants, and are suitable for popularization and application.

Description

Rice ribonuclease gene OsRNS4 and encoding protein and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and in particular relates to a rice ribonuclease gene OsRNS4 and a coding protein and application thereof.

Background

Rice is an important grain crop and has a long cultivation history. As a key object of scientific research, the disease resistance of rice is always a focus problem of great concern, and the excavation and utilization of disease resistance genes are the most economical and effective way for controlling rice diseases.

Ribonuclease (RNase) is a kind of ribonuclease widely existing in animals and plants, and its main physiological function is to control the kind and quantity distribution of intracellular RNAs, and participate in the processes of post-transcriptional cleavage, modification, degradation, etc. It has now been found that RNase in some animals has anti-tumor, immunosuppressive, angiogenesis-involved functions, and in addition, RNase is involved in the self-incompatibility, organogenesis and host defense mechanisms of some plants. Only one RNase in the plant RNase family that has been biochemically identified has other roles than general RNA degradation functions. This class of RNase family, the T2/S-RNase family, is found in fungi, bacteria, colistin and animals and plants, and is the most widely existing class of RNase known to date (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.).

In addition to S-RNases in the Solanaceae, scrophulariaceae and Rosaceae families, plants also have ribonucleases of the other RNase T2 family, called S-like RNases (S-like RNases), which are essentially identical in structure to S-RNases, but also possess different conserved amino acid residues and function differently. S-like RNases are currently found in all plant species, but their evolutionary processes and other biological roles remain unknown except that studies have found that they are not involved in self-incompatibility.

The total number of S-like RNases in rice is eight, namely OsRNS1-OsRNS8. Previous studies have found that when OsRNS4 is overexpressed in rice, the saline-alkali tolerance 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. Plantasci.2013.10.003.). However, it has not been found whether OsRNS4 is involved in the immune response process of rice, i.e., the correlation between OsRNS4 and disease resistance of rice has not been found in the prior art.

Disclosure of Invention

In order to solve the technical problems, the invention provides a rice ribonuclease gene OsRNS4 and a coding protein and application thereof.

The first object of the invention is to provide a rice ribonuclease gene OsRNS4, wherein 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.

The second purpose of the invention is to provide a protein encoded by a rice ribonuclease gene OsRNS4, and the amino acid sequence of the protein is shown as SEQ ID NO. 3.

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 OsRNS 4.

The fourth object of the present invention is to provide a transgenic rice overexpressing the ribonuclease gene OsRNS4 of said rice.

The fifth purpose of the invention is to provide the rice with the gene knocked-out rice of the rice ribonuclease gene OsRNS 4.

Preferably, overexpression of the rice ribonuclease gene OsRNS4 can enhance the resistance to false smut; knocking out the rice ribonuclease gene OsRNS4, and weakening the disease resistance of the rice to false smut.

The sixth object of the present invention is to provide a method for cultivating disease-resistant rice, comprising the steps of:

1) Transforming rice callus by using the rice ribonuclease gene OsRNS4, wherein the nucleotide sequence of an open reading frame of the rice ribonuclease gene OsRNS4 is shown as SEQ ID NO. 2;

2) Regenerating the transformed rice callus into rice;

3) Culturing regenerated rice and over-expressing the rice ribonuclease gene OsRNS 4.

The seventh object of the invention is to provide a method for detecting the expression of protein of transgenic plants of a rice ribonuclease gene OsRNS4, which comprises the following steps:

for constructed rice ribonuclease gene OsRNS4 over-expression plants: extracting leaf total protein of the transgenic plant, carrying out Western Blot (Western Blot) detection on the extracting solution, and if the target strip is detected, indicating that the plant is a transgenic positive plant, and if the target strip is not detected, indicating that the plant is a transgenic negative plant.

The eighth object of the invention is to provide a method for creating a rice OsRNS4 gene knockout material, which comprises the following steps:

1) Designing a primer according to the nucleotide sequence of the rice ribonuclease gene OsRNS4 and constructing an OsRNS4 gene knockout vector;

2) Transforming rice callus by using an OsRNS4 gene knockout vector;

3) Regenerating the transformed rice callus into rice;

4) Culturing the regenerated rice and knocking out the rice ribonuclease gene OsRNS 4.

The ninth object of the invention is to provide a gene knockout detection method of a rice ribonuclease gene OsRNS4 transgenic plant, which comprises the following steps:

for constructed knockout plants: designing a primer according to the target point, extracting leaf genome DNA of the transgenic plant, amplifying and purifying the DNA fragment by using the primer, sequencing, and if the sequencing result shows that mutation occurs and is unimodal, indicating that the transgenic plant is a transgenic positive homozygous plant.

The gene of the invention is cloned in the genome nucleotide sequence of japonica rice Japanese (Oryza sativa L. Ssp. Japonica cv. Nipponbare). According to the analysis of Japanese sunny day in NCBI, the nucleotide sequence of the OsRNS4 gene is shown as SEQ ID NO.1, the full length of the gene nucleotide is 1991bp, the gene has 3 introns and 4 exons, the CDS sections are 62-175, 891-1052, 1136-1328 and 1442-1731 respectively, and the full length of cDNA is 759bp.

Experiments prove that the disease resistance of rice to false smut can be reduced after the rice ribonuclease gene OsRNS4 is knocked out, and the balling number of the false smut is increased; after the over-expression, the disease resistance of the rice to the false smut can be improved, the balling number of the false smut is reduced, and the transgenic plant with improved resistance to the false smut can be obtained. Therefore, the ribonuclease protein and the gene thereof have important significance for cultivating the false smut-resistant plants, and are suitable for popularization and application.

The invention has the beneficial effects that:

1. the rice ribonuclease gene OsRNS4 is a good example of the participation of ribonuclease in the immune process of rice, and has certain reference value for understanding ribonuclease function and regulating plant immunity.

2. The resistance to false smut is weakened after the OsRNS4 gene is knocked out, so that the balling number of the false smut is increased; the resistance to false smut is enhanced after the over-expression, and the balling number of the false smut is reduced; therefore, the gene can be used in breeding work.

3. Since ribonucleases are a large family in plant cells, the study of the OsRNS4 gene has a good reference value for researchers to later study other ribonucleases.

Drawings

FIG. 1 shows the sequencing result of an OsRNS4 knockout transgenic strain;

FIG. 2 shows the results of protein expression detection of OsRNS4 gene over-expression transgenic lines;

FIG. 3 shows the detection of OsRNS4 mRNA level expression of an overexpressing strain

FIG. 4 shows the results of the disease course-related gene expression test of transgenic lines;

FIG. 5 shows the result of inoculating the transgenic line with rice false smut; a is an apparent observation diagram of rice ears, B is a statistical result of incidence rate, and different letters show significant differences.

Detailed Description

Unless otherwise indicated, the technical means employed are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, and the like used are all commercially available.

Example 1: osRNS4 gene knockout vector, over-expression vector construction and agrobacterium-mediated genetic transformation

1. Construction of transgenic vectors

1.1 construction of knockout vector

The nucleotide 20bp before NGG sequence in cDNA of rice ribonuclease gene OsRNS4 is selected as target design primer, and the sequence is as follows:

KO1-F：5’-aaacCATGGTATGAACTGCGAATC-3’，SEQ ID NO.4；

KO1-R：5’-ggcaGATTCGCAGTTCATACCATG-3’，SEQ ID NO.5；

KO2-F：5’-aaacACAATCCTTTCGACATTAAC-3’，SEQ ID NO.6；

KO2-R：5’-ggcaGTTAATGTCGAAAGGATTGT-3，SEQ ID NO.7。

KO1-F, KO1-R is used as a pair of primers, KO2-F, KO2-R is used as a pair of primers, and the specific construction method is as follows:

the vectors used were pOs-sgRNA, pOs-Cas9 (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 primer was first primed with ddH ₂ O was diluted to 10. Mu.M, and then equal volumes (25. Mu.l each) of forward and reverse primers were mixed and added to the PCR tube in a final volume of 50. Mu.l. Placing on a PCR instrument at 98 ℃ for 2min; the primer was allowed to form a double strand in a slow cooling down at 25℃for 10min. pOs-sgRNA was digested with Bsa I of NEB for 4h, and 0.5. Mu.l Bsa I was added at 1.5h for the initial system, which was described in the specification as 50. Mu.l. And then electrophoresis gel cutting and recycling are carried out. The ligation system was designed to take into account the concentration of recovered vector, and if the concentration of recovered vector was 30 ng/. Mu.l, the amount of recovered product was about 1.5. Mu.l per 15. Mu.l of the ligation system plus primer-attached PCR product, and the remaining volumes were pOs-sgRNA vector fragments, except for the enzyme and Buffer. The ligation was carried out overnight at 16 ℃. Transformation was screened with DH 5. Alpha. Competent, kana (kanamycin). The sequence was then recombined onto pOs-cas9 vector using LR reaction. The reaction was carried out overnight at 25 ℃. All reaction products were then transformed into DH 5. Alpha. Competent, spe (spectinomycin) for selection of positive plasmids, giving OsRNS4 knockout vector.

The primer self-ligation system is as follows:

forward primer 25. Mu.l

Reverse primer 25. Mu.l

The PCR instrument is carried out at 98 ℃ for 2min; and at 25℃for 10min.

The carrier enzyme digestion system is as follows:

pOS-sgRNA 2μg

bsa I2. Mu.l (1.5. Mu.l added first, 0.5. Mu.l added when cutting for 1.5 h)

Cutsmart Buffer 5μl

ddH ₂ O was added to the total system at 50. Mu.l and digested at 37℃for 4h.

The connection system is as follows:

the ligation was carried out overnight at 16 ℃.

The above method resulted in two knockout vectors: KO1-F, KO1-R primer constructs KO1 vector, KO2-F, KO2-R primer constructs KO2 vector.

1.2 construction of the overexpression vector

The two ends of the open reading frame nucleotide of the rice ribonuclease gene OsRNS4 are respectively intercepted to design an upstream primer and a downstream primer, and the sequence is as follows:

pC1305-3FLAG-RNS4-F：

5’-ACA GAG CTC GGT ACC ATG GAG CAG AGG AAA TTT TTG TTG-3’，SEQ ID NO.8；

pC1305-3FLAG-RNS4-R：

5’-TTT GTA GTC AAG CTT GGC CAG CAC AGT TTC AGT GG-3’，SEQ ID NO.9。

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 digested with KpnI/HindIII. Amplifying the cDNA of Japanese sunny as template to obtain the full length of the open reading frame of rice ribonuclease gene OsRNS4 (SEQ ID No.2 sequence), and connecting the digested carrier with the SEQ ID No.2 sequence segment by homologous recombination enzyme to transform colibacillus DH5 alpha. After sequencing verification, the obtained vector is the OsRNS4 gene over-expression vector, and then the plasmid is extracted, and the plasmid is subjected to heat shock and is transferred into agrobacterium EHA 105. And (3) selecting a monoclonal to expand and culture, carrying out PCR verification, adding 40% glycerol with equal volume, uniformly mixing, and preserving at-80 ℃ for later use. The vector was constructed using the high fidelity enzyme Phanta Max Super-Fidelity DNA Polymerase (Vazyme, nanjinouzan Biotechnology Co., ltd.) for amplification to reduce the possibility of base mutation in the amplified sequence. The PCR conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 60℃for 30s/kb,35 cycles; preserving at 16 ℃.

2. Genetic transformation

The genetic transformation method mediated by agrobacterium EHA105 is adopted to introduce the above OsRNS4 gene knockout vector (KO 1 vector and KO2 vector) and the OsRNS4 gene overexpression vector into japonica rice variety Japanese sunny respectively, which comprises the steps of transferring the vectors into agrobacterium strain EHA105, inducing, subculturing, pre-culturing, culturing and suspending agrobacterium, infecting and co-culturing, removing agrobacterium, screening, differentiating and transplanting, hardening and transplanting of the callus, and detecting. Reference is made to the method "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", in particular as follows:

2.1 screening of callus

The dried Japanese callus was transferred to a solid selection medium (containing 400mg/L hygromycin) for selection and incubation at 28℃for 30 days.

2.2 differentiation and transplantation

1) The selected calli were transferred to a differentiation medium containing hygromycin (50 mg/L) and cultured at 28℃for 30 days. 2) Green buds with good growth vigor are selected and transferred into rooting culture medium.

2.3 hardening off and transplanting

When the green seedlings grow to the trefoil period (about 15 days), and the root system is developed, the test tube port is opened, tap water is used for covering the surface of the culture medium, the seedlings are adapted to the external environment for 3 days, the solid culture medium at the roots of the seedlings is washed off, the seedlings are soaked in tap water for refining Miao Yizhou, and then the seedlings can be transplanted into soil.

2.4 transgenic plant Positive detection

1) The knockout transgenic plants can be sequenced at T0 generation to confirm positive homozygous lines. Taking T0 generation seedling lateral leaves, quick freezing with liquid nitrogen, grinding, extracting genome, amplifying and purifying genome fragments by using amplification primers of the gene, and then carrying out sequencing (Beijing qing biological science and technology Co., ltd.) to obtain a single-peak and mutational positive homozygous plant (figure 1).

2) The over-expression strain is obtained by taking lateral leaves of T0 generation seedlings, quick-freezing with liquid nitrogen, grinding, adding a proper amount of 5 XSDS loading buffer solution, carrying out metal bath at 100 ℃ for 10 minutes to release and denature total proteins, and preserving the samples to-80 ℃ after the completion or directly running denaturation gel. Gel electrophoresis was performed for about 2h, followed by transfer and incubation with Anti-Flag antibody (FIG. 2).

2.5 obtaining of homozygous overexpressing plants

The over-expression plants need to be screened on a hygromycin 1/2MS plate by the recovered T1 generation seeds, 3:1 character separation can be obtained, at the moment, wild type removed, homozygous and heterozygous transgenic plant seedlings are planted again and T2 generation seeds are harvested, the hygromycin 1/2MS plate is used for screening again, and the plant lines which do not have character separation are homozygous.

Example 2: RNA level expression detection of overexpressing strains

Rna extraction (kit using CWBIO, kang is century biotechnology division limited):

1) 2-3 leaves of the transgenic rice plant cultured for one week in 1/2MS are taken and transferred into a 2mL clean centrifuge tube filled with burned steel balls. 2) And (3) quick freezing by liquid nitrogen, then completely smashing the blade sample into powder by using a ball mill, adding 1mL TRIZON, vibrating for 2min, fully and uniformly mixing the sample, and standing for 5min. 3) 200. Mu.L of chloroform was added thereto, vigorously shaken for 15s, and allowed to stand at room temperature for 2 minutes. 4) Centrifuge 12000rpm at 4℃for 10min. 5) The solution in the centrifuge tube is divided into three layers, the supernatant is taken into a new RNase-free centrifuge tube, 70% ethanol prepared from RNase-free water is added into the centrifuge tube in equal volume, and the mixture is inverted and mixed uniformly. 6) Adding the mixture into an adsorption column twice, centrifuging at 12000rpm at 4 ℃ for 15s, and discarding the waste liquid. 7) 350. Mu.L RW1 was added to the column, centrifuged at 12000rpm at 4℃for 15s, and the waste liquid was discarded. 8) An incubation solution (52. Mu.L RNase-Free water+8. Mu.L 10 Xreaction buffer+20. Mu.L DNase I (1U/. Mu.L)) was prepared in advance, and after completion of the centrifugation, 80. Mu.L of the incubation solution was added dropwise to the center of the adsorption column and allowed to stand at 28℃for 15 minutes. 9) After the incubation was completed, the mixture was centrifuged at 12000rpm at 4℃for 15 seconds, and the waste liquid was discarded. 10 350. Mu.L RW1 was added, centrifuged at 12000rpm at 4℃for 15s, and the waste liquid was discarded. 11 500. Mu.L RW2 was added, centrifuged at 12000rpm at 4℃for 15s, and the waste liquid was discarded. And repeating the process once again after the completion. 12 Centrifuging with empty column at 12000rpm at 4deg.C for 2-3min, transferring the column to new RNase-free centrifuge tube, uncovering, and air drying for 3-5min.13 40. Mu.L RNase-free Water was added thereto, and the mixture was centrifuged at 12000rpm for 1min to obtain RNA, and the RNA was stored at-80℃in a refrigerator after concentration measurement.

2. Reverse transcription:

the total reverse transcription system was 20. Mu.L, which was divided into two steps

RNA 2μg

Oligo d(T)(100μM)1μL

RNA-free Water to 14μL

Mixing, placing into PCR instrument at 72deg.C for 10min, and rapidly adding into ice for 3min.

Adding into the PCR tube of the previous step

Mixing gently, placing into PCR instrument, reversing at 42deg.C for 60min, heating at 70deg.C for 10min to stop reaction, taking out to obtain cDNA solution, and immediately using or storing in-20deg.C refrigerator.

3. Fluorescent quantitative PCR:

quantitative real-time polymerase chain reaction (Quantitative real time polymerase chain reaction, qRT-PCR) experimental methods refer to FastSYBR mix, well known as century Biotech Co. The primers used were qRT-RNS4-F:5'-ATGGAAGAGCGAGTGGAAC-3' (SEQ ID NO. 10), qRT-RNS4-R:5'-GTTGTACAGTTGGTAGTCCGG-3' (SEQ ID NO. 11).

1) PCR reaction system

Reagent (20. Mu.L reaction system)

2) PCR reaction conditions

Predenaturation at 95℃for 30s

Denaturation at 95℃for 5s

Annealing/extension at 60℃for 30s

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

The quantitative result is analyzed by Bio-Rad CFX Manager, firstly, the dissolution curve, QC (quality control) and the like are analyzed, the unqualified data are removed, and the software is used for carrying out the function of Gene Study according to (1+E) ^-ΔΔ The Ct algorithm analyzes the expression of the gene of interest (FIG. 3). The result of FIG. 3 shows that the OsRNS4 gene constructed by the method has higher expression level than the wild type after being transformed.

Example 3: PR gene detection of transgenic lines

The expression of disease-associated genes is a typical experiment for detecting basic immunophenotype of plants, and we therefore detected PR genes as follows:

1. rice seed disinfection

The seeds are put into a threshing machine for peeling. The peeled seeds are moved into a sterilized 150mL triangular flask in an ultra clean workbench, 70% ethanol is added, and the mixture is gently shaken for 3min. The ethanol solution was poured off and 50% sodium hypochlorite was added at 28 ℃ +120rpm for 30min (this step was performed on a shaker). Pouring out sodium hypochlorite solution, washing with sterilized distilled water for at least 12 times, and shaking the triangular flask for 30s each time, wherein seeds are prevented from losing as much as possible when pouring out waste liquid. Spreading the washed seeds on sterilized filter paper, slightly reducing the ultra-clean typhoon, and blow-drying the seeds, but not for too long, so as to prevent the seeds from being polluted. Directly used or received into a clean 50mL tube and stored in a refrigerator at 4 ℃.

2. Exciton treatment

The sterilized seeds were spread on 1/2MS solid medium for 3 days, after which seedlings with consistent conditions were selected and transferred to 1/2MS liquid medium for further 3 days. The young rice seedlings are treated by using the exciton statin for 6 hours, then the same side leaf is taken, at least three pieces are taken for each strain, and the next experiment is carried out.

Rna extraction, reverse transcription and fluorescent quantitative PCR detection (specific procedure is as in example 2). PR gene primer is PR5-qF:5'-CAACAGCAACTACCAAGTCGTCTT-3' (SEQ ID NO. 12), PR5-qR:5'-CAAGGTGTCGTTTTATTCATCAAC-3' (SEQ ID NO. 13). The results show that the level of PR gene was significantly lower for the exciton-induced knockout strain than for the wild type, whereas the level of PR gene was significantly higher for the overexpressing strain than for the wild type (FIG. 4).

Example 4: inoculating the transgenic strain with the rice false smut

Taking out ustilaginoidea virens JS60-2 (Zheng et al 2022,Ustilaginoidea virens secretes a family of phosphatases that stabilize the negative immune regulator OsMPK6 and suppress plant immunity,Plant Cell,doi:10.1093/pl cell/koac 154.) from-80deg.C ultra-low temperature refrigerator and activating to POn the SA plate, the incubator was incubated at 28℃for 10 days. Cutting into pieces, culturing in 100mL PS culture medium at 28deg.C with shaking table at 180rpm for 5 days. Breaking hypha in the ustilago oryzae bacteria liquid to form spore hypha mixed liquid. The spore concentration was then adjusted to 1X 10 with sterile PS liquid ⁶ And each mL. 1mL of the bacterial liquid was injected into rice ears (rice ears 5 days before heading) of KO-1 (KO 1 knock-out vector transformant), KO-2 (KO 2 knock-out vector transformant), OE-1 (overexpressing vector transformant), and WT (Japanese plants, wild type) with a syringe, and 10 replicates were taken for each sample. The number and incidence of false smut were counted 21 days after inoculation.

The results showed that the knocked-out lines had significantly reduced resistance to false smut compared to the wild type, while the overexpressed lines had significantly increased resistance to false smut (fig. 5), demonstrating that the OsRNS4 gene was able to regulate the resistance of rice to false smut.

It should be noted that:

SEQ ID NO.1 sequence is as follows:

SEQ ID NO.2 sequence is as follows:

SEQ ID NO.3 sequence is as follows:

it should be noted that, when numerical ranges are referred to in the present invention, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and because the adopted step method is the same as the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. The rice ribonuclease gene OsRNS4 is characterized in that the nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.1, the open reading frame nucleotide sequence of the rice ribonuclease gene OsRNS4 is shown in SEQ ID NO.2, and the rice ribonuclease gene OsRNS4 is related to rice disease resistance.

2. The protein encoded by the rice ribonuclease gene OsRNS4 according to claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 3.

3. An expression vector comprising the nucleotide sequence of the open reading frame of rice ribonuclease gene OsRNS4 of claim 1.

4. A transgenic plant comprising the rice ribonuclease gene OsRNS4 according to claim 1.

5. The use of the rice ribonuclease gene OsRNS4 according to claim 1 for improving rice germplasm resources.

6. The use of the rice ribonuclease gene OsRNS4 according to claim 5 for improving rice germplasm resources, wherein overexpression of the rice ribonuclease gene OsRNS4 enhances resistance to false smut; knocking out the rice ribonuclease gene OsRNS4, and weakening the disease resistance of the rice to false smut.

7. The method for cultivating the OsRNS4 over-expression rice is characterized by comprising the following steps of:

1) Transferring the rice callus by using the rice ribonuclease gene OsRNS4 according to claim 1, wherein the nucleotide sequence of an open reading frame of the rice ribonuclease gene OsRNS4 is shown as SEQ ID NO. 2;

2) Regenerating the transformed rice callus into rice;

8. The method for detecting the protein expression of the transgenic plant with the rice ribonuclease gene OsRNS4 is characterized by comprising the following steps:

for rice ribonuclease gene OsRNS4 overexpressing plants constructed in claim 7: extracting leaf total protein of the transgenic plant, carrying out western blotting detection on the extracting solution, and if the target strip is detected, indicating that the plant is a transgenic positive plant, and if the target strip is not detected, indicating that the plant is a transgenic negative plant.

9. The method for cultivating the ribonuclease gene OsRNS4 knockout rice is characterized by comprising the following steps of:

1) The nucleotide sequence design primer of the rice ribonuclease gene OsRNS4 according to claim 1 and constructing an OsRNS4 gene knockout vector;

2) Transforming rice callus by using an OsRNS4 gene knockout vector;

3) Regenerating the transformed rice callus into rice;

10. The gene knockout detection method of the rice ribonuclease gene OsRNS4 transgenic plant is characterized by comprising the following steps:

for the knockout plants constructed in claim 9: designing a primer according to the target point, extracting leaf genome DNA of the transgenic plant, amplifying and purifying the DNA fragment by using the primer, sequencing, and if the sequencing result shows that mutation occurs and is unimodal, indicating that the gene knockout is successful homozygous plant.