Application of rice green revolution gene SD1 in regulation and control of rice virus resistance

Abstract

The invention relates to application of a rice green revolution gene SD1 in regulation and control of rice virus resistance. A green revolution gene SD1 in rice kasalath is knocked out through a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) technology, and an SD1 knockout transgenic material is obtained. A virus inoculation test shows that after the SD1 knockout transgenic material is infected with RRSV, SRBSDV, RGSV and RSV, the disease of the SD1 knockout transgenic material is more obvious than that of a wild type, and the infection rate of each virus is obviously higher than that of the wild type, so that the effect of the rice green revolution gene SD1 gene in regulating and controlling the rice virus resistance is disclosed. The invention provides new molecular targets and gene resources for research of rice virus resistance mechanism and breeding for disease resistance.

Description

Technical Field

[0001] The present invention relates to the fields of rice genetic engineering and virus-resistant breeding, and in particular to the application of rice green revolution gene SD1 in regulating rice virus resistance. Background Art

[0002] As one of the world's most important food crops, rice's stable yield is crucial for ensuring food security. Since the Green Revolution in the mid-20th century, semi-dwarfing breeding techniques have significantly increased rice yields. Loss of function in the rice Green Revolution gene SD1 (corresponding to GA20ox2) inhibits gibberellin biosynthesis, resulting in dwarfing of the plant, thereby enhancing lodging resistance and indirectly increasing yield. The SD1 gene primarily affects plant height by regulating gibberellin content, and its application in agricultural production has yielded significant results.

[0003] However, in recent years, rice viral diseases have posed a serious threat to rice production. Pathogens such as Rice Serrated Leaf Dwarf Virus (RRSV), Southern Rice Black-Streaked Dwarf Virus (SRBSDV), Rice Grassy Dwarf Virus (RGSV), and Rice Stripe Virus (RSV) can cause widespread rice yield reductions or even complete failure. Traditional chemical control methods suffer from environmental pollution, increased viral resistance, and high economic costs, making the development of new, environmentally friendly disease control strategies urgent.

[0004] In recent years, the role of plant hormone signaling pathways in plant disease resistance has gradually gained attention. Studies have found that gibberellins are not only involved in the regulation of growth and development, but may also play a role in the interaction between plants and pathogens. The Green Revolution gene SD1 is a key gene that regulates gibberellin synthesis. Its functional loss may lead to hormonal imbalance in the body, thereby affecting the plant's defense response. Although a large number of literatures have focused on the role of SD1 in plant dwarfing and yield improvement, its role in regulating rice virus pathogenicity has not been fully explored.

[0005] Therefore, exploring the potential regulatory role of the SD1 gene in rice virus resistance is of great significance for revealing the molecular mechanisms of rice-virus interactions and also provides new theoretical basis and technical approaches for cultivating virus-resistant rice varieties using molecular breeding methods. It is within this context that the present invention utilizes CRISPR-Cas9 technology to construct SD1 knockout materials and investigates SD1 function through a virus inoculation experimental system, aiming to provide new genetic resources and prevention and control strategies for rice virus resistance breeding. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides the application of rice green revolution gene SD1 in regulating rice virus resistance.

[0007] Specific plans include: The nucleotide sequence of the rice green revolution gene SD1 is shown in SEQ ID NO.1.

[0008] The application of the aforementioned rice Green Revolution gene SD1 in regulating rice virus resistance; Furthermore, the application is specifically: using biotechnology to knock out the rice green revolution gene SD1, thereby reducing rice virus resistance; Furthermore, the viruses include rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, rice grassy stunt virus, and rice stripe virus.

[0009] A method for regulating rice virus resistance, comprising the steps of regulating the expression of the rice green revolution gene SD1 in rice; the nucleotide sequence of the rice green revolution gene SD1 is shown in SEQ ID NO.1; Furthermore, the regulation specifically includes: knocking out the rice green revolution gene SD1 by biotechnology, thereby reducing rice virus resistance; Furthermore, the viruses include rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, rice grassy stunt virus, and rice stripe virus.

[0010] Furthermore, the method comprises the following specific steps: (1) Design a specific target based on the sequence of the rice green revolution gene SD1, embed the target sequence into an appropriate promoter element, and construct a CRISPR expression module containing the target information; (2) Integrate the CRISPR expression module into the Cas9 expression vector to construct the SD1 knockout vector; (3) Using the Agrobacterium-mediated transformation method, the SD1 knockout vector was introduced into rice to obtain transgenic rice with SD1 gene knockout.

[0011] The above method for regulating rice virus resistance is used in cultivating transgenic rice with reduced virus resistance; further, the virus includes rice sawtooth leaf dwarf virus, southern rice black-streaked dwarf virus, rice grassy stunt virus, and rice stripe virus.

[0012] The beneficial effects of the present invention are: (1) For the first time, the potential regulatory role of the rice Green Revolution gene SD1 in viral pathogenicity was revealed, providing a new perspective for in-depth analysis of the rice virus infection mechanism; (2) It provides new genetic resources and technical strategies for virus-resistant rice breeding. By regulating SD1 expression, it is expected to achieve molecular prevention and control of viral diseases. (3) The present invention adopts CRISPR-Cas9 technology, which is easy to operate, highly efficient, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : Target region sequence verification results of SD1 knockout materials.

[0014] Figure 2 : SD1 knockout reduces rice resistance to RRSV. A, Phenotypic comparison of Kasalath and its SD1 knockout material (SD1_KO) 30 days after inoculation with RRSV virus; Mock is the uninoculated control, and RRSV-infected is the inoculated group. B, qRT-PCR detection of the relative expression of the RRSV P8b gene in plants in each group 30 days after inoculation. C, Statistical analysis of the RRSV infection rate (%) of plants in each group 30 days after inoculation. ** indicates a significant difference (P < 0.01), and the error bars are standard errors (SE). Scale bar, 10 cm.

[0015] Figure 3 SD1 knockout significantly increases rice susceptibility to SRBSDV. A, Phenotypic comparison of Kasalath and its SD1 knockout lines (sd1#3, #4, and #5) 30 days after untreated (Mock) and SRBSDV inoculation. B, Statistical results of SRBSDV infection rates 6 weeks after inoculation. ** indicates a significant difference compared with the Kasalath group (P < 0.01). Error bars represent standard errors (SE).

[0016] Figure 4 SD1 knockout significantly increases the infection rate of rice to RGSV. A, Phenotypic comparison of Kasalath and its SD1 knockout lines (sd1#3, #4, and #5) 30 days after untreated (mock) and RGSV inoculation. B, Infection rate curves at various time points after RGSV inoculation. Shaded areas represent standard errors (SE). * and ** indicate statistically significant differences; ns indicates not significant.

[0017] Figure 5 SD1 knockout enhances rice susceptibility to RSV. A, Phenotypic comparison of Kasalath and its SD1 knockout lines (sd1#3, #4, and #5) untreated (mock) and 30 days after RSV inoculation. B, Infection rates at various time points after RSV inoculation. Shaded areas represent standard errors (SE). ** indicates a significant difference compared with the Kasalath group (P < 0.01). DETAILED DESCRIPTION

[0018] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0019] Example 1: Construction of SD1 knockout This example uses the CRISPR-Cas9 system to knock out the green revolution gene SD1 in rice kasalath. The specific steps are as follows: 1. Target design and primer design ① Based on the full-length sequence of the SD1 gene in rice kasalath (SEQ ID NO. 1), online CRISPR design tools (such as CRISPR-P or Benchling) were used to identify two targets with high specificity and low off-target risk, located in the first and second exons of the SD1 gene, respectively.

[0020] SD1 target 1: 5'-GACCTGAGGATGGAGCCCAA-3' SD1 target 2: 5'-TCCTCCTCCAGGACGACGT-3' ② To facilitate subsequent construction, the target sequence was fused with the gRNA framework sequence of the corresponding promoter (such as U3 and U6a), and primers for PCR amplification were designed.

[0021] Primers for SD1 target 1: U3-SD1site1-F:5'-GACCTGAGGATGGAGCCCAAgttttagagctagaaat-3' U3-SD1site1-R: 5'-TTGGGCTCCATCCTCAGGTCTgccacggatcatctgc-3' Primers for SD1 target 2: U6a-SD1site2-F: 5'-TCCTCCTCCAGGACGACGTgttttagagctagaaat-3' U6a-SD1site2-R: 5'-ACGTCGTCCTGGAGGAGGACggcagccaagccagca-3' Note: "gttttagagctagaaat" is a commonly used gRNA framework sequence, used to ensure that the constructed gRNA can fold correctly and bind to Cas9. The design of each primer ensures both specificity for the target sequence and effective splicing with the U3 or U6a promoter region.

[0022] 2. First round of PCR amplification ① Using a plasmid containing the U3 promoter sequence as a template, primers U3-SD1site1-F and U3-SD1site1-R were used to amplify the corresponding target region by PCR to obtain the U3-SD1site1 amplification product. Using a plasmid containing the U6a promoter sequence as a template, primers U6a-SD1site2-F and U6a-SD1site2-R were used to amplify the corresponding target region by PCR to obtain the U6a-SD1site2 amplification product.

[0023] ② PCR system (20 μL) includes: template DNA 1 μL, upstream primer 1 μL, downstream primer 1 μL, 2×Phanta Mix 10 μL, ddH2O 7 μL.

[0024] ③PCR program settings are: Pre-denaturation at -95°C for 3 minutes; -35 cycles: denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; Final extension at -72°C for 5 minutes.

[0025] ④ After amplification, the PCR product was confirmed to be of the expected size by agarose gel electrophoresis, and the amplified fragment was purified by gel recovery method.

[0026] 3. Construction of CRISPR Knockout Vector ① The U3-SD1site1 and U6a-SD1site2 amplification products purified from the first round of PCR were tandemly constructed. Each gRNA module was sequentially connected using a multi-round PCR splicing method.

[0027] ② Perform BsaI-HF digestion on the Cas9 expression vector (such as pC1300-Cas9) to open the vector's multiple cloning site.

[0028] ③ Mix the recovered gRNA module with the enzyme-cleaved Cas9 vector, add T4 DNA ligase, and perform the ligation reaction. The ligation system is set as follows: The total volume is 15 μL, including 70 ng of each module fragment, 100 ng of Cas9 vector, 0.5 μL of BsaI-HF, 1.5 μL of Cutsmart Buffer, 0.5 μL of T4 DNA ligase, and 0.5 μL of T4 DNA ligase buffer. The total volume is supplemented with ddH2O.

[0029] PCR ligation program: 37°C for 15 minutes, followed by 12 cycles (37°C for 5 minutes, 10°C for 5 minutes, and 20°C for 5 minutes), and finally stored at 12°C.

[0030] ④ After transformation into E. coli, single colonies were picked and the correct construction of the SD1 knockout vector was confirmed by PCR detection and sequencing.

[0031] 4. Agrobacterium-mediated transformation and material screening ①Transform the confirmed correct SD1 knockout vector into Agrobacterium (EHA105).

[0032] ②Use Agrobacterium-mediated rice tissue culture method to introduce the vector into Kasalath rice.

[0033] ③ Through tissue culture, resistance screening (such as using a screening culture medium containing appropriate antibiotics) and PCR detection, transgenic plants with SD1 gene knockout (SD1_KO) were screened out.

[0034] 5. Molecular identification of knockout results To confirm that the CRISPR / Cas9 system introduced the desired mutation at the SD1 gene target site and to assess the effects of gene knockout on expression and phenotype, the following validations were performed: Detection primers (SD1-F / SD1-R) located upstream and downstream of the target site were designed, and genomic DNA of T0 transgenic plants was amplified by PCR and sequenced by Sanger sequencing. The sequencing results detected a 2 bp deletion (-GA) at target site 2 (5'-TCCTCCTCCAGGACGACGT-3'), which resulted in a frameshift and premature stop codon ( Figure 1 ).

[0035] The specific primers are as follows: SD1-F: 5'-CGGGCAATCAATTCTAACCGT-3' SD1-R: 5'-GGTCGGTTTCTTCCCGCTT-3' Through the above steps, the SD1 gene knockout (SD1_KO) in rice kasalath, namely the SD1 knockout lines (sd1#3, #4, #5), were successfully constructed, providing a material basis for subsequent virus inoculation experiments and disease resistance evaluation.

[0036] Example 2: RRSV infection test This example systematically evaluated the differences between the SD1 knockout (SD1_KO) and wild-type kasalath after infection with rice virus through a series of virus inoculation, sample collection, molecular detection and phenotypic analysis experiments, thereby verifying the role of SD1 in regulating rice resistance to RRSV virus.

[0037] 1. Material Preparation and Seedling Cultivation ① Select healthy rice seeds for wild-type kasalath and SD1_KO transgenic materials respectively.

[0038] ② Pre-treat the seeds: soak in 70% ethanol for 10 minutes, then treat with 0.1% mercuric chloride solution for 30 minutes, and then rinse with plenty of sterile water.

[0039] ③ Sow the seeds in a nutrient solution and incubate for two weeks to promote seedling growth. Each group has 40 plants, and the experiment was repeated three times to ensure reproducibility.

[0040] 2. Virus Inoculation ① Virus source: Use laboratory-verified rice viruses, specifically Rice Serrated Leaf Dwarf Virus (RRSV).

[0041] ② Carrier insects: Use non-toxic brown planthoppers, which first feed on rice plants containing the target virus for 3 to 5 days to allow them to acquire the virus.

[0042] ③Inoculation method: a. Transfer infected insects (2 to 3 per plant) to the plants to be tested and maintain contact for 3 days to ensure virus transmission.

[0043] b. After inoculation, remove the insects from the plants using sterile methods and transfer the plants to a culture environment containing nutrient soil.

[0044] c. Continue culturing at a constant temperature (30°C) with 16 hours of light / 8 hours of darkness for 30 days.

[0045] 3. Virus Detection and Molecular Analysis (1) Sample collection and RNA extraction a. 30 days after inoculation, approximately 0.5 g of leaves were randomly collected from each group of plants and immediately frozen in liquid nitrogen.

[0046] b. Use TRIzol reagent to extract total RNA according to the instructions and test the concentration and purity.

[0047] (2) cDNA synthesis cDNA was synthesized using a standard reverse transcription kit with 0.5 μg of total RNA as a template. The reaction conditions were as described in the kit instructions.

[0048] (3) Real-time quantitative PCR (qRT-PCR) detection qRT-PCR was used to detect viral RNA levels, and rice EF-1α was used as the internal reference gene.

[0049] The specific primers are as follows: Internal reference gene primers: OsEF-1α-F: 5'-ACATTGCCGTCAAGTTTGCTG-3' OsEF-1α-R: 5'-AACAGCCACCGTTTGCCTC-3' RRSV detection primers: RRSV-P8b-F: 5'-TTGGAGCTGGAGTGAACGTC-3' RRSV-P8b-R: 5'-GCTACTGTGTAAGTGGCGGT-3' The PCR reaction system (20 μL) includes: 2×RealStar Green Fast Mixture: 10μL cDNA template (diluted): 2 μL Forward primer (10 μM): 0.5 μL Reverse primer (10 μM): 0.5 μL Add ddH2O to 20 μL The PCR program settings were: Pre-denaturation at -95°C for 3 minutes; - 35 cycles: 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds; Final extension at -72°C for 5 minutes.

[0050] The relative expression of viral RNA was calculated using the 2^(-ΔΔCt) method.

[0051] 4. Phenotypic Observation and Symptom Assessment ① 30 days after virus inoculation, conduct a comprehensive observation of the plants in each group and record symptoms such as the distribution of lesions, plant dwarfing, and yellowing of leaves.

[0052] ② Calculate the incidence rate (number of infected plants / total number of plants) and disease severity score for each group of plants, and take representative images.

[0053] ③ Statistical analysis was performed on the molecular detection data and phenotypic observation results to verify the effect of SD1 knockout on the degree of viral infection.

[0054] 5. Results Analysis and Discussion To clarify the role of rice SD1 gene in virus resistance, this study used Kasalath variety and its SD1 knockout material (SD1_KO) as background materials, inoculated with rice serrated leaf dwarf virus (RRSV), and systematically compared the differences in phenotype, virus accumulation and infection rate between the two. Figure 2 As shown in A, when not inoculated (Mock), both Kasalath and SD1_KO plants grew robustly, while SD1_KO plants showed a dwarf phenotype. After RRSV inoculation, SD1_KO plants showed more severe dwarfing and shrinkage compared to Kasalath plants, and their growth potential was significantly weakened, indicating that SD1 knockout reduced rice resistance to RRSV. To further quantitatively analyze the amount of virus accumulation, the expression level of the RRSV P8b gene was detected by qRT-PCR ( Figure 2 B). The results showed that after RRSV inoculation, the relative expression of P8b in the SD1_KO group was significantly higher than that in the Kasalath group, suggesting that knocking out SD1 significantly promoted the replication and accumulation of RRSV virus in the plant. At the same time, the RRSV infection rate of the two groups of plants after inoculation was compared ( Figure 2 C), the Kasalath group showed approximately 55% susceptibility, while the SD1_KO group showed a significant difference of approximately 80%. Statistical analysis further confirmed that SD1 knockout increased susceptibility to RRSV in rice. These results suggest that the rice SD1 gene plays an important role in resistance to RRSV infection, and that SD1 knockout significantly reduced rice resistance to RRSV, leading to higher viral accumulation and more severe disease phenotypes.

[0055] Example 3: SRBSDV, RGSV and RSV infection assay To systematically evaluate the role of the SD1 gene in rice virus resistance, this example used Kasalath and its SD1 knockout lines (sd1#3, #4, and #5) as materials and inoculated them with southern rice black-streaked dwarf virus (SRBSDV), rice grassy stunt virus (RGSV), and rice stripe virus (RSV), respectively. The material preparation, virus inoculation, sample collection, and molecular detection methods were the same as in Example 2, with the only difference being the viruses and primers used.

[0056] The specific primers are as follows: SRBSDV detection primers: SRBSDV-S8-F:5'-GAGCTTCAGTGAATTGGAAAC-3' SRBSDV-S8-R:5'-AGAGCCACACAATTATTTAAAATATGT-3' RGSV detection primers: RGSV-CP-F: 5'-AATGGTATGATTGGCTTA-3' RGSV-CP-R: 5'-ATAAGAAGAAGGCAGAAG-3' RSV detection primers: RSV-CP-710F: 5'-GACAAGAAGAAGGAAGAT-3' RSV-CP-810R: 5'-ATCGTATTGACAGACATA-3' like Figure 3 、 Figure 4 、 Figure 5 As shown in the results, after inoculation with the three viruses, although Kasalath showed certain symptoms, the SD1 knockout lines all showed more severe typical virus infection symptoms such as plant dwarfing, leaf yellowing, wilting and even death.

[0057] Infection rate statistics showed that under the infection of three viruses, SRBSDV, RGSV, and RSV, the infection rates of SD1 knockout materials (sd1#3, #4, #5) were significantly higher than those of Kasalath (P<0.01), and the differences persisted at all time points. Specifically, 6 weeks after SRBSDV and RGSV inoculation, the infection rates of SD1 knockout materials reached approximately 60-80% and 60-90%, respectively, much higher than the approximately 20-40% of the Kasalath group; 8 weeks after RSV inoculation, the infection rate of SD1 knockout materials was also significantly higher than that of the control. The overall results show that SD1 gene knockout generally weakened rice's resistance to a variety of important viruses and significantly increased susceptibility.

Claims

1. Rice Green Revolution Gene SD1 , characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

2. The rice green revolution gene according to claim 1 SD1 Application in regulating virus resistance in rice.

3. The use according to claim 2, characterized in that: Using biotechnology to knock out rice's Green Revolution genes SD1 , thereby reducing rice virus resistance.

4. The use according to claim 2, characterized in that: The viruses include rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, rice grassy stunt virus, and rice stripe virus.

5. A method for regulating virus resistance in rice, characterized by: Including regulating the rice green revolution genes in rice SD1 The steps of expression; The rice green revolution gene SD1 The nucleotide sequence is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that: Using biotechnology to knock out rice's Green Revolution genes SD1 , thereby reducing rice virus resistance.

7. The method according to claim 5, characterized in that: The viruses include rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, rice grassy stunt virus, and rice stripe virus.

8. The method according to claim 6, wherein: The specific steps are as follows: (1) Based on the rice green revolution gene SD1 Sequence design of specific targets and embedding of target sequences into appropriate promoter elements to construct a CRISPR expression module containing target information; (2) Integrate the CRISPR expression module into the Cas9 expression vector to form SD1 Knockout vector; (3) Using Agrobacterium-mediated transformation method, SD1 The knockout vector was introduced into rice to obtain SD1 Gene knockout transgenic rice.

9. Use of the method according to any one of claims 5 to 8 in cultivating transgenic rice with reduced virus resistance.

10. The use according to claim 9, characterized in that: The viruses include rice serrated leaf dwarf virus, southern rice black-streaked dwarf virus, rice grassy stunt virus, and rice stripe virus.

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