Application of phytolectin coding gene OsChtBL1 in resisting rice stripe virus

Abstract

The invention relates to a phytolectin coding gene in rice and application of the phytolectin coding gene in rice stripe virus resistant rice breeding. According to the invention, through a genetic transformation technology, the phytolectin gene OsChtBL1 is successfully introduced into wild Nipponbare rice, so that a transgenic line with stable inheritance is obtained. After the OsChtBL1 gene is overexpressed in rice, an artificial rice stripe virus inoculation test is carried out, and the result shows that compared with wild Nipponbare rice, the rice with the overexpressed OsChtBL1 gene shows higher disease resistance.

Description

Technical Field

[0001] The invention relates to the field of transgenic technology and plant disease prevention and control, and in particular to a rice lectin gene OsChtBL1 and an application thereof in resistance to rice stripe virus. Background Art

[0002] Rice is one of the most important food crops in the world and the staple food for about half of the world's population. The infection and spread of insect-borne viruses usually cause rice to suffer dual damage from insect vectors and insect-borne viruses. Rice stripe virus (RSV), as an important virus that is widely distributed in my country's rice-growing areas and extremely harmful, poses a serious threat to my country's rice production safety. RSV is a representative member of the genus Tenuivirus. Its virus particles are in the form of non-enveloped filaments with a diameter of about 3-9nm. RSV is a type of multi-partite negative-sense single-stranded RNA virus. Its genome consists of 4 single-stranded RNA fragments and encodes 7 proteins (Xu et al., 2021). In the natural state, RSV can infect more than 80 species of grass plants, including foxtail grass, barley, millet, corn, sorghum and oats, in addition to important food crops such as rice, wheat, barley, millet, corn, sorghum and oats. Under laboratory conditions, RSV can infect dicotyledonous plants such as Nicotiana benthamiana and Arabidopsis thaliana through mechanical inoculation. In the early stages of RSV, the heart leaves of rice will show chlorotic stripes or patches. The period from the seedling stage to the tillering stage of rice is the most serious period of RSV disease. After the disease occurs, the main manifestations are curling and softening of the heart leaves, yellowing of the leaves and the formation of intermittent light yellow stripes. The plants become short and develop slowly. When the disease is serious, the heading will not appear and the diseased plants will die directly, posing a huge threat to the safety of rice production in my country.

[0003] In the long-term struggle with herbivorous insects and pathogens in natural habitats, plants have gradually evolved a complex and effective defense network. Plant lectins are a group of proteins that can recognize various carbohydrates based on their molecular structure and biochemical properties but do not catalyze them. They are widely present in many plant species. Based on amino acid sequence similarity, molecular structure analysis, and the types of carbohydrates they bind, sixteen plant lectin families containing lectin domains have been identified. These families are usually named after the species in which the lectin of this family was first discovered. Some major families include the Galanthus nivalis agglutinin (GNA) family, the jackfruit lectin (jacalin) family, the lysine M (LysM) domain lectin family, and the hevein domain lectin family. In recent decades, with the advancement of high-throughput sequencing technology, the role of plant lectins is no longer limited to carbohydrate recognition. Molecular structure determination and omics analysis have provided deeper insights into the functions of plant lectins, revealing their involvement in multiple key biological processes. However, only a limited number of lectin families have been studied in depth. For example, GNA lectins have a high affinity for mannose in insect gastrointestinal epithelial cells, leading to insect poisoning. LysM domain lectins act as receptors for chitin oligosaccharides from pathogens or symbionts, triggering innate immunity mediated by plant pathogen-associated molecular patterns (PAMPs) and microbe-associated molecular patterns (MAMPs).

[0004] Protein folding is a critical and complex process in biology, involving the spontaneous formation of functional three-dimensional structures from amino acid chains. This process is driven primarily by changes in entropy and energy rather than being directly directed by DNA or RNA. For secretory proteins, they are synthesized as unfolded polypeptides in the endoplasmic reticulum (ER) and fold with the assistance of factors within the ER. The ER quality control (ERQC) system is responsible for detecting improperly folded proteins and degrading them through the ER associated degradation (ERAD) system to prevent these misfolded proteins from causing damage to the cell. Under conditions of environmental stress or high secretory demand, the demand for protein folding may exceed the processing capacity of the ER, leading to endoplasmic reticulum stress (ER stress). This stress triggers the unfolded protein response (UPR), a homeostatic mechanism that reduces the burden of unfolded proteins by adjusting the protein folding and degradation capacity of the ER. Studies of the UPR in plants have shown that it is characterized by the upregulation of genes related to protein folding and ERAD. In addition, long-term ERstress may promote autophagy and lead to cell death in some cases (Howell., 2013). Recent studies have found that multiple marker genes related to ER stress are upregulated in the early stages of plant immune response, indicating that plant immunity may require enhanced ER function. These research results emphasize the importance of protein folding and endoplasmic reticulum stress response in maintaining plant protein homeostasis and cellular processes. The present invention identified a plant lectin protein in rice that can activate endoplasmic reticulum stress-mediated antiviral immunity, providing an important molecular mechanism and potential breeding target for breeding new rice varieties with RSV resistance. Summary of the invention

[0005] The invention relates to a rice lectin encoding gene OsChtBL1 and an encoded protein thereof.

[0006] In one aspect, the OsChtBL1 gene has a nucleotide sequence as shown in SEQ ID NO:1.

[0007] On the other hand, the amino acid sequence of the protein encoded by the OsChtBL1 gene is shown in SEQ ID NO:2.

[0008] In some embodiments, the CDS sequence of the rice lectin encoding gene OsChtBL1 (SEQ ID NO: 1) is as follows:

[0009] ATGACCATGACGTCGACGACGACGAAGGCCATGGCGATGGCGGCGGCGGTCCTCGCCGCCGCCGCCGTCGCGGCGACGAACGCGCAGACGTGCGGGAAGCAGAACGACGGCATGATCTGCCCGCACAACCTGTGCTGCAGCCAGTTCGGGTACTGCGGCCTCGGCCGCGACTACTGCGGCACGGGGTGCCAGAGCGGCGCCTGCTGCTCCAGCCAGCGCTGCGGCAGCCAGGGCGGCGGCGCCACCTGCTCCAACAACCAGTGCTGCAGCCAGTACGGCTACTGCGGCTTCGGCTCCGAGTACTGCGGCTCCGGGTGCCAGAACGGGCCGTGCCGCGCCGACATCAAGTGCGGCCGCAACGCCAACGGCGAGCTCTGCCCCAACAACATGTGCTGCAGCCAGTGGGGATACTGCGGCCTCGGCAGCGAGTTCTGCGGCAACGGATGCCAGAGCGGCGCGTGCTGCCCGGAGAAGCGGTGCGGCAAGCAGGCCGGCGGGGACAAGTGCCCCAACAACTTCTGCTGCAGTGCCGGCGGCTACTGCGGCCTCGGCGGCAACTACTGCGGCTCCGGCTGCCAGAGCGGCGGCTGCTACAAGGGTGGCGACGGCATGGCGGCCATCCTGGCTAACAACCAGAGCGTCTCTTTCGAAGGGATCATCGAGTCAGTGGCTGAGCTTGTGTAG。

[0010] The amino acid sequence (SEQ ID NO: 2) of the rice plant lectin-encoding gene OsChtBL1 is as follows:

[0011] MTMTSTTTKAMAMAAAVLAAAAVAATNAQTCGKQNDGMICPHNLCCSQFGYCGLGRDYCGTGCQSGACCSSQRCGSQGGGATCSNNQCCSQYGYCGFGSEYCGSGCQNGPCRAD IKCGRNANGELCPNNMCCSQWGYCGLGSEFCGNGCQSGACCPEKRCGKQAGGDKCPNNFCCSAGGYCLGGGNYCGSGCQSGGCYKGGDGMAAILANNQSVSFEGIIESVAELV*.

[0012] On the other hand, the present invention relates to the use of the rice lectin encoding gene OsChtBL1 in the breeding of Tenuivirus-resistant Gramineae food crops.

[0013] In some embodiments, the Tenuivirus is preferably Rice stripe virus (RSV) or Maize stripe virus (MSpV); most preferably Rice stripe virus (RSV).

[0014] In some embodiments, the gramineous food crops are preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Nipponbare.

[0015] In another aspect, the present invention relates to a method for preparing a rice stripe virus resistant transgenic plant, the steps comprising:

[0016] 1) Construction of OsChtBL1 overexpression vector;

[0017] 2) Rice genetic transformation, Agrobacterium transformation and callus induction culture

[0018] 3) Identification of positive transgenic plants;

[0019] In some embodiments, the step of constructing the OsChtBL1 overexpression vector further comprises:

[0020] Specific primers were designed according to the sequence of OsChtBL1 shown in SEQ ID No: 1, and the full-length sequence of OsChtBL1 was cloned from the cDNA of wild-type Nipponbare rice, and gel recovery was performed after PCR amplification; the pCAMBIA1300 vector containing the MYC tag was digested with BamHI and SacI, and the products recovered from the gel were connected to the vector respectively, and the positive clones were detected by PCR technology, and sent to a biological company for sequencing, confirming that the pCAMBIA1300-OsChtBL1 expression vector with the MYC tag was successfully constructed.

[0021] In some embodiments, the steps of rice genetic transformation, Agrobacterium transformation and callus induction culture further include:

[0022] Induction of callus: Carefully select mature rice seeds and undergo a strict disinfection procedure to induce callus formation. The seeds are disinfected in 75% ethanol for 1 minute, then rinsed with sterile water, disinfected in 30% sodium hypochlorite for 20 minutes, and finally rinsed thoroughly with sterile water 5 to 6 times. The disinfected seeds are transferred to the induction medium and cultured in a light incubator at 28°C for 3 weeks to promote the growth of callus. Subsequently, the formed callus is transferred to the subculture medium with sterile tweezers and cultured under the same conditions for another week;

[0023] Transformation and cultivation of Agrobacterium: The plasmid containing the target vector was transformed into the Agrobacterium tumefaciens strain GV3101 by electroporation. This process involves mixing 5 μL of plasmid with 100 μL of Agrobacterium competent cells, then electroporating at 220V in a pre-cooled electrode cup at 4°C, followed by shaking culture in non-resistant LB liquid medium for 3 hours, and finally spreading on LB solid medium containing Kan and Rif, and culturing in the dark at 28°C for 2 days until single colonies appear;

[0024] Screening and infection of Agrobacterium: Use PCR technology to identify the above Agrobacterium, pick positive clones and culture them overnight in a constant temperature shaking incubator at 28°C. Then select an appropriate amount of callus tissue, put it in a sterile triangular flask, add an appropriate amount of Agrobacterium suspension, and infect it at room temperature for 20 minutes, shaking it from time to time to ensure uniform contact. After the infection is completed, pour out the bacterial solution, place the callus tissue on sterile filter paper to absorb excess bacterial solution, and then transfer it to a solid co-culture medium covered with sterile filter paper, and culture it in the dark at 26°C for 3 days;

[0025] Callus culture and rooting: After co-cultivation, the callus was washed and finally rinsed with sterile water containing carbenicillin. The callus was blown dry in the clean bench for about 30 minutes, then transferred to the screening medium containing hygromycin B and cultured in the dark at 28-30°C for 3-4 weeks to screen out successfully transformed cells. Finally, when the seedlings grew to 2-3cm and showed obvious roots, they were transferred to the rooting medium to promote further growth. Rooting culture was carried out under sterile light conditions at 28-30°C.

[0026] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0027] The wild-type Nipponbare rice and transgenic rice overexpressing OsChtBL1-MYC were extracted using denaturing lysis buffer, and rice Rubisco was used as an internal reference protein. Immunoblotting analysis was performed using an antibody that can specifically recognize the MYC tag. Among them, the target band of OsChtBL1-MYC could be detected in the two transgenic rice lines OsChtBL1-OE#1 and OsChtBL1-OE#6 overexpressing OsChtBL1, but not in the wild-type Nipponbare rice, indicating that the transgenic rice line was successfully constructed.

[0028] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0029] The expression levels of endoplasmic reticulum stress (ER stress) related genes in wild-type Nipponbare rice and transgenic rice overexpressing OsChtBL1 were detected by RT-qPCR technology. Rice OsUBQ5 was used as an internal reference gene, and the quantitative primers are shown in SEQ ID No: 7-10. The relative expression levels of ERstress related genes in the two transgenic rice lines overexpressing OsChtBL1, OsChtBL1-OE#1 and OsChtBL1-OE#6, were significantly higher than those in wild-type Nipponbare rice.

[0030] In some embodiments, the step of identifying positive transgenic plants further comprises:

[0031] The resistance of transgenic lines overexpressing OsChtBL1 to rice stripe virus was tested by artificial RSV inoculation experiment.

[0032] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, pH=5.8.

[0033] In some embodiments, the subculture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, pH=5.8.

[0034] In some embodiments, the rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH=5.8.

[0035] In some embodiments, the co-culture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, pH=5.2.

[0036] In some embodiments, the denaturing lysis buffer comprises: 6.0 ml 10% SDS, 1.0 ml 1.0 M Tris-HCl (PH=6.8), 0.2 ml β-mercaptoethanol, and 2.8 ml ddH2O.

[0037] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0038] 1) The present invention constructs an overexpression vector of a plant lectin encoding gene OsChtBL1 in rice, and introduces it into the wild-type Nipponbare rice variety by Agrobacterium infection, thereby obtaining two transgenic rices with stable inheritance, which are named OsChtBL1-OE#1 and OsOsChtBL-OE#6. The successful construction of the OsChtBL1 transgenic line was proved by immunoblotting analysis technology, and the artificial inoculation RSV experiment confirmed its resistance to rice stripe virus, which provides a new direction for the cultivation of transgenic rice with RSV resistance and adds valuable genetic resources to the rice germplasm resource bank.

[0039] 2) The present invention uses RT-qPCR technology to detect the expression level of endoplasmic reticulum stress (ER stress) related genes in transgenic lines overexpressing OsChtBL1 and analyze the resistance to rice stripe virus. It is found that OsChtBL1, a plant lectin in rice, can effectively activate ER stress, which greatly enriches the research direction of rice disease resistance theory.

[0040] 3) The present invention studies the mechanism of action of a plant lectin encoding gene OsChtBL1 in rice during RSV infection, thereby discovering the interaction between rice and the virus, laying a foundation for further theoretical research on disease resistance and breeding of resistant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1: Protein accumulation levels of OsChtBL1-MYC in wild-type Nipponbare rice and transgenic rice overexpressing OsChtBL1.

[0042] Figure 2 :Relative expression levels of endoplasmic reticulum stress (ERstress)-related genes in wild-type Nipponbare rice and transgenic rice overexpressing OsChtBL1.

[0043] Figure 3 : Pathogenesis symptoms of wild-type Nipponbare rice and transgenic rice overexpressing OsChtBL1 after RSV infection.

[0044] Figure 4 : Detection of virus content in wild-type Nipponbare rice and transgenic rice overexpressing OsChtBL1 after RSV infection.

[0045] mock: healthy rice; RSV: rice infected with rice stripe virus (RSV); NIP: wild-type Nipponbare rice. DETAILED DESCRIPTION

[0046] The rice varieties used in this series of experiments are: Nipponbare

[0047] The relevant culture medium components are as follows:

[0048] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, PH=5.8.

[0049] Subculture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: 15HBZ0601) 24.1 g / L, 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, PH = 5.8.

[0050] Co-culture medium: N6 culture medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, pH = 5.2.

[0051] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6) 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH = 5.8.

[0052] Denaturing lysis buffer: 6.0 ml 10% SDS, 1.0 ml 1.0 M Tris-HCl (PH=6.8), 0.2 ml β-mercaptoethanol, 2.8 ml ddH2O.

[0053] Example 1: Construction of OsChtBL1 overexpression vector in rice

[0054] 1) Cloning of Rice OsChtBL1 Gene

[0055] Primers OsChtBL1-F and OsChtBL1-R were designed according to the sequence of OsChtBL1 (SEQ ID No: 1), and the primer sequences are as follows:

[0056] OsChtBL1-F:ATGACCATGACGTCGACGACGACG (SEQ ID NO: 3);

[0057] OsChtBL1-R:TTACACAAGCTCAGCCACTGACT (SEQ ID NO:4);

[0058] The total volume was 50 μL, including 2×Phanta Max Buffer 25 μL, 1.5 μL each of upstream and downstream primers (10 μM), 1 μL of dNTPMix (10 mM), 1 μL of cDNA template, 1 μL of Phanta Max Super-Fidelity DNA Polymerase, and 16.5 μL of ddH2O.

[0059] PCR amplification program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 3 min, 35 cycles; final extension at 72°C for 10 min.

[0060] The PCR product was recovered, connected to the pMD18-T vector (Takara Biotech, catalog number D101A), cloned, and sent for testing to confirm that the pMD18-T-OsChtBL1 recombinant plasmid was successfully obtained.

[0061] 2) Construction of OsChtBL1 overexpression vector

[0062] Primers for connecting restriction sites were designed to construct the OsChtBL1 gene into the binary expression vector pCAMBIA1300 (Beijing Dingguo Changsheng Biotechnology Co., Ltd., MCV033). After the pCAMBIA1300 vector was digested with BamHI and SacI, the PCR products were connected to the vectors respectively. Positive clones were selected for sequencing. The primer sequences used are as follows:

[0063] Lic-OsChtBL1-F:

[0064] CGACGACAAGACCGTCACCATGACCATGACGTCGACGACGACG (SEQ ID NO: 5);

[0065] Lic-OsChtBL1-R:

[0066] GAGGAGAAGAGCCGTCGCACAAGCTCAGCCACTGACT (SEQ ID NO: 6);

[0067] Sequencing confirmed that the pCAMBIA1300-OsChtBL1 expression vector had been successfully constructed.

[0068] Example 2: Rice genetic transformation

[0069] 1) Callus induction: Carefully select mature rice seeds and undergo a strict sterilization procedure to induce callus formation. Seeds are sterilized in 75% ethanol for 1 minute, then rinsed with sterile water, sterilized in 30% sodium hypochlorite for 20 minutes, and finally rinsed thoroughly with sterile water 5 to 6 times. The sterilized seeds are transferred to the induction medium and cultured in a light incubator at 28°C for 3 weeks to promote the growth of callus. Subsequently, the formed callus is transferred to the subculture medium with sterile forceps and cultured under the same conditions for another week.

[0070] 2) Transformation and cultivation of Agrobacterium: The plasmid containing the target vector was transformed into the Agrobacterium tumefaciens strain GV3101 by electroporation. This process involves mixing 5 μL of plasmid with 100 μL of Agrobacterium competent cells, then electroporating at 220V in a 4°C precooled electrode cup, followed by shaking culture in a non-resistant LB liquid medium for 3 hours, and finally spreading on LB solid medium containing Kan and Rif, and culturing in the dark at 28°C for 2 days until single colonies appear.

[0071] 3) Screening and infection of Agrobacterium: The above Agrobacterium was identified by PCR technology, and the positive clones were picked and cultured overnight in a constant temperature shaking incubator at 28°C. Then, an appropriate amount of callus tissue was selected, placed in a sterile triangular flask, and an appropriate amount of Agrobacterium suspension was added. It was infected at room temperature for 20 minutes, and it was shaken from time to time to ensure uniform contact. After the infection was completed, the bacterial solution was poured out, and the callus tissue was placed on sterile filter paper to absorb the excess bacterial solution, and then transferred to a solid co-culture medium covered with sterile filter paper, and cultured in the dark at 26°C for 3 days.

[0072] 4) Callus culture and rooting: The callus after co-cultivation is washed and finally rinsed with sterile water containing carbenicillin. The callus is blown dry in the clean bench for about 30 minutes, then transferred to a screening medium containing hygromycin B and cultured in the dark at 28-30°C for 3-4 weeks to screen out successfully transformed cells. Finally, when the seedlings grow to 2-3 cm and show obvious roots, they are transferred to rooting medium to promote further growth. Rooting culture is carried out under sterile light conditions at 28-30°C.

[0073] Example 3: Identification of positive transgenic plants

[0074] 1) Extraction of total protein from rice plants

[0075] Metamorphosis lysis buffer was used to extract the total protein of wild-type Nipponbare rice and positive OsChtBL1 overexpressing transgenic rice grown normally in an artificial climate chamber for about 15 days.

[0076] 2) Immunoblotting analysis to detect the protein accumulation level of OsHDA706-MYC

[0077] Immunoblotting analysis was performed using the rice total protein extracted as described above, with rice Rubisco as an internal reference gene and an antibody that can specifically recognize the MYC tag used to detect the accumulation level of OsHDA706-MYC in wild-type Nipponbare rice and positive overexpression OsChtBL1 transgenic rice.

[0078] The test results are as follows Figure 1 As shown, the protein accumulation level of OsChtBL1-MYC in the two transgenic lines OsChtBL1-OE#1 and OsChtBL1-OE#6 overexpressing the OsChtBL1 gene was significantly higher than that in the control group, proving that the transgenic rice lines were successfully constructed.

[0079] Example 4: Relative expression levels of ER stress-related genes in transgenic rice overexpressing OsChtBL1

[0080] TRIzol reagent was used to extract the leaves of wild-type Nipponbare rice and positive OsChtBL1 overexpressing transgenic rice grown normally in an artificial climate chamber for about 15 days. The extracted RNA was reverse transcribed into cDNA using III qRT SuperMix (manufacturer: Novozyme, catalog number: R323-01). The above cDNA samples were used with the OsUBQ5 gene of rice as an internal reference. The relative expression levels of ER stress-related genes were as follows Figure 2 The quantitative primer sequences are as follows:

[0081] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:7);

[0082] OsUBQ5-R:ACGCCTAAGCCTGCTGGTT(SEQ ID NO:8);

[0083] OsPDIL2-3-F:GCTCCACTCCGTAGTGCTTT(SEQ ID NO:9);

[0084] OsPDIL2-3-R:AACCTCACCATCTTTGCCGT(SEQ ID NO:10);

[0085] OsBiP1-F:CGGTATCGAGACTGTCGGTG(SEQ ID NO:11);

[0086] OsBiP1-R:GTCCTCAGCCTTCACGTTCA(SEQ ID NO:12);

[0087] OsIRE1-F:GGACTTCCTTCGGCACAACT(SEQ ID NO:13);

[0088] OsIRE1-R:ACGACCATGACGAAGCTGTT(SEQ ID NO:14);

[0089] OsSDF2-1-F:CTTGGCGGCAAAACCAGAAA(SEQ ID NO:15);

[0090] OsSDF2-1-R:CGCCAACACCACATACCTCT(SEQ ID NO:16);

[0091] OsERDJ3B-F:AGCACCACATGAGCGATTCA(SEQ ID NO:17);

[0092] OsERDJ3B-R:TCGTCGGTTAGGGTTTTCGG(SEQ ID NO:18);

[0093] OsBzip50-F:TGAGCATCGTGTGCCTACTC(SEQ ID NO:19);

[0094] OsBzip50-R:GGAACGGTCCGGTATCTAGC(SEQ ID NO:20);

[0095] OsSAR1-F:GGACTACTACGCCCAGGTTG (SEQ ID NO:21);

[0096] OsSAR1-R:CGAGGTAGTAGCACAGCTCC (SEQ ID NO: 22);

[0097] OsFes1C-F: TACAGATTGGGCGTGCAGTT (SEQ ID NO: 23);

[0098] OsFes1C-R:TGTGCCCTCTTTGAGCTTGT (SEQ ID NO: 24);

[0099] The results are as follows Figure 2 As shown, the relative expression levels of endoplasmic reticulum stress-related genes in the two transgenic lines OsChtBL1-OE#1 and OsChtBL1-OE#6 overexpressing OsChtBL1 were significantly higher than those in the wild-type Nipponbare rice, indicating that OsChtBL1 activated the endoplasmic reticulum stress response mechanism of rice.

[0100] Example 5: Artificial inoculation of RSV

[0101] 1) The wild-type Nipponbare rice and two transgenic lines overexpressing OsChtBL1, OsChtBL1-OE#1 and OsChtBL1-OE#6, were soaked at 37°C for 2-3 days. After the seeds germinated, seeds with consistent germination levels were selected and evenly sown in 1 L glass beakers (at least 3 replicates, 30 seedlings in each replicate), and cultured in an artificial climate chamber at 30°C with a light cycle of 16 h light and 8 h dark.

[0102] 2) The SBPHs were artificially reared to 1-2 years old, then infected on RSV-infected rice seedlings for 3-5 days and then transferred to healthy rice seedlings for the transition period (the time required for RSV to complete the infection cycle in the SBPH, 7-15 days).

[0103] 3) RSV-carrying or RSV-free wild-type Nipponbare rice and two transgenic lines overexpressing OsChtBL1, OsChtBL1-OE#1 and OsChtBL1-OE#6, were inoculated at a ratio of 3 SBPHs per seedling, and all insects were swept out 3 days after inoculation (RSV-carrying and healthy SBPHs of the same age were used for virus inoculation experiments).

[0104] 4) 30 days after transplanting the infected rice materials into the field, the disease symptoms of the plants were observed and the disease status of the rice was determined by qRT-PCR.

[0105] Example 6: Resistance analysis of transgenic lines overexpressing OsChtBL1

[0106] The disease symptoms were observed 30 days after the rice was transplanted into the field. Compared with the wild-type Nipponbare rice, the two transgenic lines overexpressing OsChtBL1, OsChtBL1-OE#1 and OsChtBL1-OE#6, showed obvious resistance to RSV. Figure 3 As shown. The total RNA of rice material was extracted as shown in Example 3, reverse transcribed into cDNA, and the OsUBQ5 gene of rice was used as an internal reference. The expression level of RSV CP gene (virus capsid protein encoding gene) was further detected by qRT-PCR, as shown. Figure 4 As shown, the expression level of RSV CP gene in the two transgenic lines overexpressing OsChtBL1 was significantly lower than that in wild-type Nipponbare rice; the above results indicate that overexpressing OsChtBL1 in rice can significantly enhance rice resistance to RSV infection. The quantitative primer sequences are as follows:

[0107] qRT-CP-F: AGGCAATCAATGACATCTCC (SEQ ID NO: 25);

[0108] qRT-CP-R: ATCTCTCACAAAGCCAGTGC (SEQ ID NO: 26);

[0109] In summary, the experimental results show that compared with wild-type Nipponbare rice, transgenic rice plants overexpressing OsChtBL1 can activate the endoplasmic reticulum stress response in rice and enhance the resistance of rice to RSV infection. Therefore, the present invention successfully obtains RSV infection-resistant transgenic rice through transgenic means, adds valuable genetic resources to the rice germplasm resource bank, is of great significance to agricultural development and national food security, and has achieved unexpected technical effects.

Claims

1. Use of a plant lectin encoding gene OsChtBL1 gene in rice breeding resistant to rice stripe virus (RSV), wherein the nucleotide sequence of the OsChtBL1 gene is shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the amino acid sequence of the OsChtBL1 gene is shown in SEQ ID NO:

2.

3. A method for preparing rice stripe virus-resistant transgenic rice, comprising the steps of: (1) Construction of rice expression vector Specific primers were designed according to the OsChtBL1 nucleotide sequence provided by SEQ ID NO: 1, and the full-length sequence of OsChtBL1 was cloned from rice cDNA; amplification was performed by PCR reaction system for gel recovery, and then the pCAMBIA1300 vector containing the MYC tag was digested with BamHI and SacI, and the PCR product was connected to the vector, positive clones were screened, and sequencing was performed after amplification and culture, and finally the pCAMBIA1300-OsChtBL1 expression vector was confirmed to be constructed; (2) Genetic transformation of rice Callus culture: Select mature rice seeds, carefully peel off the husks, and transfer them to a centrifuge tube; then, sterilize the seeds with 75% ethanol for 1 minute; Pour off the ethanol and rinse the seeds once with sterile water to remove the residual ethanol; place the seeds in a 30% sodium hypochlorite solution for 20 minutes; after the sodium hypochlorite treatment, rinse the seeds again with sterile water for 5 to 6 times to remove as much residual disinfectant as possible; then, use a pipette to remove excess water in the centrifuge tube and transfer the seeds to the pre-prepared induction medium; place the medium in a 28°C light incubator for 3 weeks to promote the formation of callus tissue; after the callus tissue is formed, use pre-sterilized tweezers to transfer the callus tissue to the subculture medium, and continue to culture it in a 28°C light incubator for 1 week to promote further growth and differentiation of the callus tissue; Transformation and culture of Agrobacterium: Use a pipette to take 5 μL of the plasmid containing the target vector and add it to 100 μL of Agrobacterium tumefaciens GV3101 competent cells; then, use a pipette to gently blow to ensure that the plasmid and bacterial cells are fully mixed; the mixed bacterial suspension is transferred to a sterile electroporation cup pre-cooled at 4°C, and electroporation transformation is performed at a voltage of 220V; after the electroporation transformation is completed, non-resistant LB liquid culture medium is added to the electroporation cup, and the bacterial suspension is shaken and cultured in a constant temperature shaker at 28°C for about 3 hours to promote bacterial recovery and plasmid expression; the cultured bacterial suspension is evenly spread on LB solid culture medium containing 50 μg / ml kanamycin (Kan) and 50 μg / ml rifampicin (Rif) to screen successfully transformed bacteria; these culture dishes are then placed in a dark environment at 28°C for 2 days until single colonies appear; Agrobacterium transfection of callus tissue: single clones were selected from the mature single colony plates for amplification and PCR verification. Positive clones were inoculated into 100 ml LB liquid medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in a constant temperature shaker at 28°C at 220 rpm until OD 600 = about 0.6; add the Agrobacterium suspension to the triangular flask containing rice callus and co-culture at room temperature for 20 minutes to promote the infection of Agrobacterium to the callus; after the infection, pour out the bacterial solution and use sterile filter paper to gently absorb the excess bacterial solution on the callus; transfer the treated callus to a solid co-culture medium covered with a layer of sterile filter paper and culture it in a dark environment at 26°C for 3 days; Cultivation and rooting of callus: After 3 days of cultivation, use a 1ml blue pipette tip to transfer the callus from the co-culture medium to a sterilized triangular flask; first rinse twice with sterile water, and finally rinse with sterile water containing 500μl / L carbenicillin; after using a pipette to remove excess water, place the callus on sterile filter paper and air dry it in a sterile environment of a clean bench for 30 minutes; after the surface water of the callus is completely dry, transfer it to the screening medium and culture it in a dark environment at 28-30℃ for 3-4 weeks to screen the successfully transformed callus; when the callus differentiates into seedlings and grows to 2-3cm, and the root system is obvious, transfer these seedlings to the rooting medium and culture them in a sterile light environment at a temperature of 28-30℃ to ensure the healthy growth of the seedlings and the stable development of the root system; (3) Identification of positive transgenic plants Denaturing lysis buffer was used to extract total proteins from positive transgenic plants for immunoblot analysis, and rice Rubisco was used as an internal reference protein. The protein accumulation level of OsChtBL1-MYC in the two transgenic lines OsChtBL1-OE#1 and OsChtBL1-OE#6 overexpressing the OsChtBL1 gene was significantly higher than that in the control group, proving that the transgenic rice line was successfully constructed.

4. The preparation method according to claim 3, wherein the induction medium comprises: 24.1 g / L N6 medium, 2 mg / L 2,4-D, and pH = 5.

8.

5. The preparation method according to claim 3, wherein the subculture culture medium comprises: 24.1 g / L N6 culture medium, 2 mg / L 2,4-D, 50 mg / L hygromycin, 300 mg / mL cephalosporin, and pH = 5.

8.

6. The preparation method according to claim 3, wherein the rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, and pH = 5.

8.

7. The preparation method according to claim 3, wherein the co-culture medium comprises: 24.1 g / L N6 culture medium, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, and pH = 5.

2.

8. The preparation method according to claim 3, wherein the denaturing lysis solution comprises: 6.0 ml 10% SDS, 1.0 ml 1.0 M Tris-HCl (PH=6.8), 0.2 ml β-mercaptoethanol, and 2.8 ml ddH2O.

9. A method for activating endoplasmic reticulum stress in rice, comprising transferring a rice lectin OsChtBL1 gene into rice, wherein the nucleotide sequence of the OsChtBL1 gene is shown in SEQ ID NO:

1.

10. A method for improving the resistance of rice to rice stripe virus, comprising introducing a rice lectin OsChtBL1 gene into wild-type Nipponbare rice, wherein the nucleotide sequence of the OsChtBL1 gene is shown in SEQ ID NO: 1.

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