Application of OsFEI1 protein and coding gene thereof in regulating and controlling resistance of plant to waterstraw dwarf virus

Abstract

The invention relates to the technical field of agricultural biology, and particularly discloses application of OsFEI1 protein and a coding gene thereof in regulation of resistance of plants to waterstraw dwarf viruses, the amino acid sequence of the OsFEI1 protein is shown as SEQ ID NO: 1, or the amino acid sequence of the OsFEI1 protein has 95%, 96%, 97%, 98% or more than 99% of identity with the amino acid sequence shown as SEQ ID NO: 1, and the coding gene of the OsFEI1 protein has a nucleotide sequence shown as SEQ ID NO: 2. The OsFEI1 gene is knocked out, the amino acid sequence of the OsFEI1 gene, which is derived from the sequence and has biological functions, is basically reserved, and experiments prove that the sensitivity of the rice with the OsFEI1 gene knocked out through a CRISPR / Cas9 tool to RGSV is remarkably enhanced. It shows that the OsFEI1 gene plays an important role in the anti-RGSV process of the rice, and normal expression of the OsFEI1 gene is essential for maintaining the resistance of the rice to the RGSV. The invention provides a new clue for deeply understanding a molecular interaction mechanism between the rice and the RGSV, and also provides a potential gene target for cultivating a new variety of anti-RGSV rice.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to application of OsFEI1 protein and its encoding gene in improving plant resistance to rice grassy stunt virus. Background Art

[0002] Rice, one of the most important food crops in my country and around the world, has long been attacked by a variety of pathogens, resulting in reduced yields. Rice viruses, often called "rice cancer," include Rice Grassy Stunt Virus (RGSV), a member of the genus Tenuivirus in the family Bunyaviridae. First discovered in the Philippines in 1963, RGSV has been identified as a member of the genus Tenuivirus in the family Bunyaviridae. Its genome consists of six negative-sense single-stranded RNA strands. RGSV is transmitted by the insect vector, the brown planthopper, through a persistent multiplication mechanism, independent of eggs, seeds, pollen, soil, or mechanical damage. Once infected, the virus rapidly reproduces within the planthopper, further spreading to its host plant, rice. Symptoms of rice infected with RGSV include significant dwarfing, weed-like growth, increased tillering, chlorotic and yellowing of leaves, narrow leaf shape, and the formation of brown rust-like spots (similar to lesions). Severe cases can lead to fruit failure or even death. Effective control strategies for this disease remain unavailable. Currently, there are no reports on the role of the OsFEI1 protein and its encoding gene in rice resistance to the virus. Summary of the Invention

[0003] The main purpose of the present invention is to provide an OsFEI1 protein or its encoding gene for regulating plant resistance to rice grassy stunt virus ( Rice Grassy Stunt Virus ), the amino acid sequence of the OsFEI1 protein is as shown in SEQ ID NO: 1, or has 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as shown in SEQ ID NO: 1, and substantially retains the amino acid sequence of the biological function derived from the sequence.

[0004] Those skilled in the art will appreciate that a protein sequence can be modified without altering its function by replacing, substituting, adding, or deleting one or more amino acids in the sequence. Therefore, the present invention should be understood to include such modifications to the amino acid sequence shown in SEQ ID NO: 1.

[0005] The nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 2, or has 95%, 96%, 97%, 98% or 99% or more identity with the nucleotide sequence shown in SEQ ID NO: 2, and substantially retains the nucleotide sequence of the biological function derived from the sequence.

[0006] OsFEI1 The base sequence of the gene is not limited to that shown in SEQ ID NO: 2 in the sequence listing, but also includes a DNA sequence having any codon selected and combined corresponding to each amino acid residue in OsFEI1 and the modified OsFEI1 protein. The codon selection can be performed according to conventional methods or with reference to the codon preference of the host.

[0007] In one embodiment, the present invention provides a recombinant vector or plasmid for use in regulating plant resistance to rice grassy stunt virus, wherein the recombinant vector contains a nucleotide sequence as shown in SEQ ID NO: 2, or a nucleotide sequence having 95%, 96%, 97%, 98% or 99% or more identity with the nucleotide sequence as shown in SEQ ID NO: 2, and substantially retains the biological function derived from the sequence.

[0008] In one embodiment, the present invention provides an application of an engineered bacterium in regulating plant resistance to rice grassy stunt virus, wherein the engineered bacterium contains the recombinant vector or plasmid.

[0009] In one embodiment, the present invention provides a kit for use in regulating plant resistance to rice grassy stunt virus, wherein the kit contains the recombinant vector or plasmid, or the engineered bacteria.

[0010] In another embodiment, the present invention provides the use described above, wherein knocking out the OsFEI1 protein or its encoding gene reduces the resistance of a plant to rice grassy stunt virus.

[0011] In some embodiments of the use, the plant is a monocot or a dicot, preferably rice.

[0012] In one embodiment, the present invention provides a method for cultivating a rice grassy stunt virus-resistant plant, which overexpresses or normally expresses the OsFEI1 protein or its encoding gene, wherein the amino acid sequence of the OsFEI1 protein is shown in SEQ ID NO: 1.

[0013] In one embodiment, the present invention provides the use of OsFEI1 protein or its encoding gene as a molecular marker in rice virus-resistant molecular marker-assisted breeding, the amino acid sequence of the OsFEI1 protein is shown in SEQ ID NO: 1, and the virus is rice grassy stunt virus.

[0014] In one embodiment, the present invention provides a method for screening plants resistant to rice grassy stunt virus, detecting the expression level of OsFEI1 protein or its encoding gene. If the OsFEI1 protein or its encoding gene is overexpressed or normally expressed, the plant has stronger resistance to rice grassy stunt virus. The amino acid sequence of the OsFEI1 protein is shown in SEQ ID NO: 1.

[0015] The technical effects achieved by the present invention are: The present invention has demonstrated through experiments that when the CRISPR / Cas9 gene editing technology is used to knock out the OsFEI1 gene in rice, the experimental results show that the rice becomes more sensitive to RGSV infection, that is, the infection ability of RGSV is promoted. This shows that the OsFEI1 gene plays an important role in the resistance of rice to RGSV, and its normal expression is essential for maintaining rice resistance to RGSV. The present invention provides new clues for a deeper understanding of the molecular interaction mechanism between rice and RGSV, and also provides potential gene targets for breeding new RGSV-resistant rice varieties. In the future, we can further study the specific molecular regulatory mechanism of the OsFEI1 gene in the process of rice resistance to RGSV, and explore how to optimize the expression of the OsFEI1 gene through genetic engineering technology, thereby improving rice resistance to RGSV and providing a strong guarantee for stable and high rice yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For transfer OsFEI1 Western identification of rice; Figure 2 For transfer OsFEI1 Identification diagram of CRISPR / Cas9 rice; Figure 3 For transfer OsFEI1 and OsFEI1 qRT-PCR detection of RGSV virus-related genes in CRISPR / Cas9 rice and wild-type rice (Zhonghua 11) after RGSV infection; Figure 4 For transfer OsFEI1 and OsFEI1 Statistical graph of the incidence of CRISPR / Cas9 rice and wild-type rice (Zhonghua 11) after RGSV infection; Figure 5 Symptoms of different rice lines infected with RGSV.

[0017] In the figure of the present invention, ZH11 represents the wild type Zhonghua 11 rice. FEI1 OE stands for OsFEI1 Gene overexpression in rice, fei1 represent OsFEI1 Knockout rice. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available. The present invention will be described in detail using the following examples, but these examples are not intended to limit the present invention.

[0019] Example 1 OsFEI1 Overexpression vector construction 1. Obtaining the OsFEI1 protein and its encoding gene The amino acid sequence of rice OsFEI1 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2. They are publicly available in the Rice Genome Annotation Database (http: / / rice.plantbiology.msu.edu / ) and can be retrieved using LOC_Os12g44090. OsFEI1 Design PCR primers based on the DNA sequence starting with the ATG initiation codon of the CDS sequence: OsFEI1 CDS-F: 5'-ATGGACAGCACAAGCAACAAGA-3', OsFEI1 CDS-R: 5'-TTAGAGTCCAGGTGCTGCTC-3'.

[0020] 2. Cloning of the full-length sequence of OsFEI1 and obtaining a recombinant vector containing this fragment Primers were designed based on the OsFEI1 sequence and the required restriction enzyme sites were added at both ends of the primers. The primer sequence was OsFEI1-5' Sfi I: 5'- GGCCATTACGGCCATGGACAGCACAAGCAACAAGA-3', OsFEI1-3' Sfi I: 5'-GGCCGAGGCGGCCTTAGAGTTCCAGGTGCTGCTC-3'.

[0021] According to the instructions, TRIzol Reagent from Invitrogen was used to extract the rice cultivar Zhonghua 11 ( Oryza sativa L. japonica The specific process is as follows: Xu Yu et al. "Rice gluten protein of Zhonghua 11" Gt 1. Gene cloning and wax gene promoter-directed Gt1 Construction of Gene Expression Vectors," Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204; cDNA was obtained by reverse transcription using the company's SuperScript II reverse transcriptase. The primer used for reverse transcription was a 16-nucleotide Oligod (T) primer.

[0022] The cDNA obtained by reverse transcription was used as a template and the above gene specific primers OsFEI1-5' Sfi I and OsFEI1-3' Sfi I was used for PCR (Polymerase Chain Reaction) reaction to obtain a 2391 bp PCR product containing the OsFEI1 gene sequence.

[0023] After the PCR product was recovered, restriction enzyme Sfi I was digested to recover the 1887 bp PCR product with sticky ends; the vector pCambia2300 was digested with restriction endonucleases Sfi I enzyme digestion, the vector backbone was recovered; the above 1887 bp PCR product with sticky ends was ligated to the vector backbone using T4 ligase, and transformed into E. coli strain DH5α to obtain transformants. The plasmid extracted from the transformant was sent for sequencing, and the plasmid was the OsFEI1 gene inserted into the vector pCambia2300. Sfi The vector obtained between the restriction enzyme I sites was named pCambia2300-MYCFEI1 ， That is the OsFEI1 overexpression recombinant vector.

[0024] Example 2, CRISPR / Cas9 OsFEI1 Gene knockout vector construction pH-Ubi-cas9-fei1:sgRNA: According to the website ftp: / / ftp.cbi.pku.edu.cn / pub / supplementary_file / . According to the PAM site of the terminal sequence of NGG, select the target OsFEI1 The 20bp specific target sequence at the N-terminus of the coding region is: CCATGGACAGCACAAGCAACT. Primers are designed according to the DNA sequence shown in the target sequence, and primers are added at both ends of the primers. Bsa I restriction enzyme cutting site, the primer sequence is: OsFEI1-5'BsaI: 5'-GGCATGCGATGGACAGCACAAGCAACAAGA-3', OsFEI1-3' Bsa I: 5'-AAACAGACTTAGAGTTCCAGGTGCTGCTC-3'; The two primers were annealed and then cleaved with restriction enzymes Bsa I digested the intermediate vector pOs-sgRNA, recovered the digestion product, and then connected the primer annealing product to the intermediate vector digestion product with T4 DNA ligase to generate an intermediate vector containing the target sequence. The above-mentioned ligation product was transformed into Escherichia coli strain DH5α, coated with Cannabinoid resistance medium, and colony PCR screening obtained positive transformants. The positive transformants and the plasmid of the final vector pH-Ubi-cas9-7 were extracted separately, and the two were recombined in a 1:1 ratio using the LR enzyme in the Gateway system. The Escherichia coli strain DH5α was transformed and coated with spectinomycin-resistant medium to obtain transformants. The plasmid of the transformant was extracted and sent for sequencing. The positive transformant was the final OsFEI1 knockout recombinant vector, named pH-Ubi-cas9-fei1:sgRNA.

[0025] 2. Obtaining Rice with Overexpression and Knockout of OsFEI1 1) Callus induction culture Hull the Zhonghua 11 rice seeds and soak them in 70% ethanol for 10 minutes, then in 0.1% mercuric chloride for 30 minutes to sterilize the surface. Rinse the seeds with plenty of sterile water to remove any surface solution, then remove any moisture with sterile filter paper. Place the seeds on a plate containing mature embryo callus induction medium, seal the edges with Parafilm, and incubate in a dark room at 26°C. After approximately 15 days, carefully remove the grown callus and transfer it to mature embryo subculture medium, continuing incubation under the same conditions. Subculture should be repeated every two weeks. For transformation, select callus that has been subcultured for approximately five days and is pale yellow, granular, and present.

[0026] 2) Cultivation of Agrobacterium pCambia2300-MYCFEI1 and pH-Ubi-cas9-fei1:sgRNA were electroporated into Agrobacterium EHA105 to obtain the recombinant bacteria EHA105 / pCambia2300-MYCFEI1 and pH-Ubi-cas9-fei1:sgRNA.

[0027] Streak EHA105 / pCambia2300-MYCFEI1 and pH-Ubi-cas9-fei1:sgRNA on LB plates containing antibiotics (50 mg / L Kanamycin, 50 mg / L Rifampicin) and culture at 28°C for 2 days. Pick a single colony and inoculate it into liquid LB medium and culture it at 28°C with shaking until the OD 600 The concentration of acetosyringone was about 0.5, and acetosyringone was added to a final concentration of 100 mM to obtain an Agrobacterium suspension for transforming rice callus tissue.

[0028] 3) Co-culture of rice callus and Agrobacterium Place the subcultured callus into a sterile Erlenmeyer flask and pour the Agrobacterium suspension into the flask until it is submerged. Incubate at room temperature for 20 minutes, gently shaking the flask occasionally to ensure full contact between the callus and the bacterial suspension. Gently remove the callus with sterile tweezers, place it on sterile filter paper to absorb excess bacterial suspension, and transfer it to a co-cultivation medium plate lined with sterile filter paper. Incubate in the dark at 28°C for 3 days to obtain the co-cultivated callus.

[0029] 4) Screening and differentiation of resistant callus The co-cultivated callus tissue was washed with an appropriate amount of sterile water to remove any residual Agrobacterium on the surface. The callus was then placed on a screening medium and incubated in the dark at 26°C for screening. After two weeks, the callus was transferred to a fresh screening medium and screened for another two weeks. Calli that appeared to be in good condition after two rounds of screening were selected and transferred to differentiation medium plates. The cells were incubated in the dark for three days before being transferred to a light incubator (15 hours per day) for light incubation. Differentiated seedlings were visible after one month. When the differentiated seedlings reached approximately 2 cm in size, they were transferred to rooting medium in a conical flask and incubated for approximately two weeks. Seedlings with good growth and a well-developed root system were selected, the culture medium removed from the roots with tap water, and the cells were transplanted into soil. The seeds were harvested to obtain T1 generation rice seeds with overexpression and knockout of the OsFEI1 gene. These seeds were then sown to obtain T1 generation rice with overexpression and homozygous knockout of the OsFEI1 gene.

[0030] T1 rice seeds were initially screened with hygromycin or G418 (the pCambia2300 vector carries a G418 resistance selection gene, while the pH-Ubi-cas9-7 vector carries a hygromycin resistance selection gene). Germinated seeds indicated that the vector had been transferred into the rice plants. Germinated seeds were planted in soil and allowed to grow for two weeks. 0.1 g of leaves were then collected and ground into a powder using liquid nitrogen.

[0031] OsFEI1 Add 200 μl of protein extraction buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) to the leaf powder of the over-expressed transgenic rice line, incubate on ice for 10 minutes, boil at 100°C for 10 minutes, centrifuge at 12,000 rpm at 4°C for 10 minutes, take the supernatant, perform SDS-PAGE, transfer to the membrane, and detect by Western blotting. SDS-PAGE and Western Blot were performed according to the known methods and product instructions. The antibody used was anti-MYC-HRP (Sigma), and the antibody anti-Actin was used to detect the endogenous Actin protein of rice as an internal reference. Figure 1The ones with a band at 45 KDa were positive, indicating that the gene was transferred and the protein was expressed. Two strains #01 and #02 were selected for subsequent disease resistance analysis experiments.

[0032] OsFEI1 Genomic DNA was extracted from leaf powder of the knockout transgenic rice lines using the High-Efficiency Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No. DP350). 0.5 g of genomic DNA was then used as a template for PCR reactions using primers FEI1-F': 5'-ACCCTCACTCTCACTCCCAC-3' and FEI1-R': 5'-ATGTAAGGGAGATTTCTGGTAG-3'. The PCR products were directly sequenced and then aligned. Figure 2 As shown, DNA sequencing results showed that the two positive strains were named OsFEI1 #01, OsFEI1 #02, among which, OsFEI1 #01 is missing bases 1 to 13. OsFEI1 #02 lacks bases 2 to 10, causing a frameshift and premature termination of the OsFEI1 amino acid sequence, leading to the loss of the OsFEI1 protein.

[0033] Example 3 OsFEI1 Overexpression of rice had no significant effect on RGSV infection, and knockout OsFEI1 Promote RGSV infection.

[0034] 1) Identification of RGSV infection by quantitative RT-PCR (qRT-PCR) to identify the expression of RGSV CP Using brown planthoppers carrying RGSV (the pathogen is rice dwarf virus ( Rice Grassy Stunt Virus )) were inoculated with T1 generation OsFEI1 Thirty plants of each type of rice, including overexpression, knockout, and wild-type rice, were inoculated and cultured at 30 degrees Celsius during the day, 22 degrees Celsius at night, and 60% humidity. Five brown planthoppers were inoculated on each plant. The brown planthoppers were caught after three days of feeding, and the fed rice were cultured in a sunlit greenhouse (natural light, temperature. The experiment was repeated three times, and the results were averaged).

[0035] Four weeks after exposure to the virus, the T1 generation OsFEI1 Rice leaf powder from overexpressing, knockout, and wild-type rice Zhonghua 11 strains was added to Trizol (Invitrogen) and RNA was extracted according to the manufacturer's instructions. Genomic DNA from the RNA was then digested with RQ1 DNase (Promega, Cat. No. M610A) according to Table 1 below: Table 1 shows the digestion system Then, 2 μg of digested RNA was used for reverse transcription qRT-PCR. For detailed methods, refer to Invitrogen M-MLV Reverse Transcriptase (Cat. No. 28025-021). OsEF1a was used as an internal reference, and the primers for the internal reference were: EF1a-F: 5'-GCACGCTCTTTCTTGCTTTCACTCT-3' EF1a-R: 5'-AAAGGTCACCACCATACCAGGCTT-3', Detect the expression level of RGSV CP. The primers for CP are: CP-F: 5'-AGAGCAGTTTCCTGTAGTCCC-3', CP-R: 5'- CCAGTTCGGCTGTTCAGATTAG-3'.

[0036] The results are as follows Figure 3 As shown, it can be seen that OsFEI1 Overexpression of T1 rice ( FEI1 OE) had little difference in CP accumulation compared with wild-type rice Zhonghua 11. OsFEI1 Knockout rice ( fei1 ) was significantly higher than that of wild-type rice Zhonghua 11 (the asterisks in the figure indicate significant differences). OsFEI1 Overexpression is not particularly sensitive to RGSV infection. When CRISPR / Cas9 gene editing technology is used to modify the OsFEI1 After the gene was knocked out, experimental results showed that rice became more sensitive to RGSV infection.

[0037] 2) Determine RGSV infection rate by phenotype Four weeks after infection, 30 T1 transgenic plants were observed. OsFEI1 Overexpression, 30 strains OsFEI1 Symptoms of CRISPR / Cas9 transgenic rice and 30 wild-type rice plants (those infected with RGSV showed dwarfing, increased tillering, and yellow and elongated leaves, while those not infected with RGSV showed no dwarfing, increased tillering, and yellow and elongated leaves). Figure 4 As shown, the number of symptomatic plants was counted and the virus infection rate was calculated as (number of phenotypic plants / total number of plants)*%.

[0038] The results are summarized in Table 2: Table 2 shows the statistical results of virus infection rate of transgenic rice after virus infection As can be seen from Table 2, compared with the wild type Zhonghua11 and overexpression OsFEI1 Compared with knockout OsFEI1 The rice with the gene has a higher susceptibility to disease. In addition, we took photos of different strains of susceptible rice, such as Figure 5 Shown are healthy animals 4 weeks after infection. OsFEI1 Overexpression and OsFEI1 The disease symptoms of knockout rice. It can be seen that the disease symptoms of knockout rice are different from those of wild type Zhonghua11 and overexpression OsFEI1 Rice comparison OsFEI1 Knockout rice has stronger disease symptoms, with a higher degree of dwarfism and significantly yellowed and elongated leaves ( Figure 5 ).

[0039] In summary, when the OsFEI1 The discovery of a gene that promotes RGSV infection could provide a good target for virus-resistant rice breeding, preventing viral infection at the root and stabilizing rice yields.

[0040] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Various modifications and changes can be made to the details based on all the teachings that have been published. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. OsFEI1 protein or its encoding gene regulates plant resistance to rice grassy stunt virus ( Rice Grassy Stunt Virus ) resistance, characterized in that, The amino acid sequence of the OsFEI1 protein is as shown in SEQ ID NO: 1, or has 95%, 96%, 97%, 98% or 99% or more identity with the amino acid sequence as shown in SEQ ID NO: 1, and substantially retains the amino acid sequence of its biological function derived from the sequence.

2. The use according to claim 1, characterized in that The nucleotide sequence of the encoding gene is as shown in SEQ ID NO: 2, or has 95%, 96%, 97%, 98% or 99% or more identity with the nucleotide sequence as shown in SEQ ID NO: 2, and substantially retains the nucleotide sequence of the biological function derived from the sequence.

3. Use of a recombinant vector or plasmid in regulating plant resistance to rice grassy stunt virus, characterized in that: The recombinant vector contains a nucleotide sequence as shown in SEQ ID NO: 2, or a nucleotide sequence that has 95%, 96%, 97%, 98% or 99% or more identity with the nucleotide sequence as shown in SEQ ID NO: 2, and substantially retains the biological function derived from the sequence.

4. Use of an engineered bacterium in regulating plant resistance to rice grassy stunt virus, characterized in that: The engineered bacteria contains the recombinant vector or plasmid described in claim 3.

5. Use of a kit for regulating plant resistance to rice grassy stunt virus, characterized in that: The kit contains the recombinant vector or plasmid described in claim 3, or the engineered bacteria described in claim 4.

6. The use according to any one of claims 1 to 5, characterized in that: Knocking out the OsFEI1 protein or its encoding gene reduces the plant's resistance to rice grassy stunt virus.

7. The use according to claim 6, characterized in that The plant is a monocotyledonous plant or a dicotyledonous plant, preferably rice.

8. A method for cultivating rice grassy stunt virus-resistant plants, characterized in that: The OsFEI1 protein or its encoding gene is overexpressed or normally expressed, and the amino acid sequence of the OsFEI1 protein is shown in SEQ ID NO:

1.

9. Use of OsFEI1 protein or its encoding gene as a molecular marker in rice virus-resistant molecular marker-assisted breeding, characterized in that: The amino acid sequence of the OsFEI1 protein is shown in SEQ ID NO: 1, and the virus is rice strawy stunt virus.

10. A method for screening plants resistant to rice grassy stunt virus, characterized in that: The expression level of OsFEI1 protein or its encoding gene is detected. Overexpression or normal expression of OsFEI1 protein or its encoding gene indicates stronger resistance to rice grassy stunt virus. The amino acid sequence of the OsFEI1 protein is shown in SEQ ID NO: 1.

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