Rice light system I subunit OsPsaL gene and application thereof in resisting rice stripe virus and southern rice black-streaked dwarf virus

Abstract

The invention relates to a rice light system I subunit OsPsaL gene which is subjected to targeted editing by utilizing a CRISPR / Cas9 technology and application of the rice light system I subunit OsPsaL gene in resisting rice stripe disease and southern rice black-streaked dwarf virus.

Description

Technical Field

[0001] The present invention relates to the field of transgenic technology and plant disease prevention and control, and in particular to targeted editing of the rice photosystem I subunit OsPsaL gene using CRISPR / Cas9 technology and its application in resistance to rice viruses. Background Art

[0002] Rice stripe virus (RSV), also known as rice stripe leaf blight virus, belongs to the genus Tenuivirus and is transmitted by the gray planthopper (Laodelphax striatellus). RSV is a negative-sense single-stranded RNA (-ss RNA) virus. Its genome is named RNA1, RNA2, RNA3 and RNA4 in descending order of molecular weight, encoding 7 proteins, each of which has a different function to promote viral infection and replication. Among them, RNA1 encodes a 337kDa RNA-dependent RNA polymerase (RNA-dependent RNA polymerase, RdRp) on the complementary strand, which mainly assists in viral replication. The other three RNAs each have a bidirectional coding strategy, with the open reading frame at the 5' end of the RNA encoding proteins P2-P4 and the proteins pc2-pc4 on the complementary strand. PC2 participates in vector transmission by overcoming insect barriers, and the P2 protein functions as a silencing suppressor; PC3 is a nucleocapsid protein and is also believed to play a key role in the transovarial transmission of rice stripe virus; P3 is a silencing suppressor that competes with host proteins for double-stranded and single-stranded siRNA to prevent the formation of silencing complexes; PC4 (32 kDa) is mainly distributed near or within the cell wall and is considered to be a movement protein; P4 is a non-structure-specific protein.

[0003] Rice stripe leaf blight is a viral disease spread by the small brown planthopper. Once the rice is infected, it has a great impact on the yield. And it is very difficult to prevent and control. Generally, the yield loss of the affected fields is 20%-30%, and serious fields will cause a total crop failure. At present, the disease has become a major outbreak disease in Jiangsu, Henan and other provinces. It is an important viral disease of rice in my country with a wide distribution area, severe occurrence and great harm. Therefore, the prevention and control of rice stripe leaf blight is related to the national economy and people's livelihood, and it is very necessary to take effective prevention and control measures; so far, the most economical and effective measure to prevent and control rice stripe leaf blight is to cultivate disease-resistant varieties. Therefore, the cultivation of transgenic rice resistant materials can provide important theoretical and technical guidance for crop disease-resistant breeding.

[0004] In recent years, southern rice black-streaked dwarf virus (SRBSDV) has attracted widespread attention as a virus that seriously threatens rice production. The virus is highly latent and extremely destructive, and is difficult to effectively prevent and control. Southern rice black-streaked dwarf virus (SRBSDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Fijivirus under the family Reoviridae. SRBSDV is a non-enveloped icosahedral virus whose genome consists of 10 double-stranded RNA fragments. In recent years, phylogenetic tree analysis has shown that the gene sequence homology between SRBSDV and Rice black-streaked dwarfvirus (RBSDV) is very high. By analogy with RBSDV, researchers found that SRBSDV P5-1, P6, and P9 are important components of the virus replication toxin. Among them, the P6 protein can coordinate virus infection and transmission by interacting with OsRTH2, a key transcription factor in the ethylene signaling pathway. P7-2 interacts with S-phase kinase-associated protein 1 (OsSKP1) to form an SCF complex. P8 protein encodes the inner capsid protein of the virus, which has the function of inhibiting transcriptional activity and inhibits the antiviral defense response mediated by it by interacting with the rice auxin pathway transcription factor OsARF17. P10 encodes the outer shell protein, which can stimulate the endoplasmic reticulum stress response. A variety of strategies have been adopted in the research on SRBSDV, including screening disease-resistant varieties, improving cultivation techniques, and using biological control agents. In addition, the use of transgenic technology to develop disease-resistant rice is also one of the research hotspots, although it is still in the laboratory stage. Therefore, further in-depth and systematic research is needed to strengthen scientific research and explore the biological characteristics and infection mechanism of the virus in order to effectively prevent and control SRBSDV and ensure the stability and sustainable development of rice production.

[0005] Photosynthesis is the main process by which higher plants convert sunlight into chemical energy. The light reactions of photosynthesis are driven by photosystems I and II (PSI and PSII). PSI is a membrane protein complex that, as an integral part of the photosynthetic machinery, enables sunlight-driven transmembrane electron transfer. As a component of PSI, PsaL is essential for the formation of PSI trimers, a process that may depend on the binding of calcium ions to the PsaL subunit. However, based on current research, the relationship between PsaL and the antiviral mechanism of rice has not been studied in detail.

[0006] Based on the defects of the prior art, the inventors of the present invention used the CRISPR / Cas9 gene editing system to target OsPsaL, constructed a knockout vector of YL-Hu-OsPsaL, and transformed the callus formed by the mature embryo induction of rice Nipponbare NIP by rice callus transfection technology. After obtaining T0-generation transgenic rice seeds, they continued to multiply to obtain T1-generation seeds, extracted DNA from young leaves of T1-generation single plants, amplified sgRNA targeting sites with target-specific primers, and sequenced them to obtain stable inherited homozygous mutant transgenic plants. Summary of the invention

[0007] The present invention relates to targeted editing of the OsPsaL gene of the rice photosystem I subunit using CRISPR / Cas9 technology.

[0008] In one aspect, the nucleotide sequence of the OsPsaL gene is shown in SEQ ID NO.1.

[0009] .

[0010] On the other hand, the amino acid sequence of rice photosystem I subunit OsPsaL (SEQ ID NO: 2) is as follows: MATAYAPMASQLMKSSLVCSKPRGLSGASLTRRPRFTVKAIQSEKPTYQVVQPINGDPFIGSLETPVTSSPLVAWYLSNLPAYRTAVSPLLRGIEVGLAHGYLLVGPFALTGPLRNTPVHGQAGALGAAGLVAILSVCLTMYGVASFGEGEPSTAPTLTLTGRKKEADKLQTADGWAKFTGGFFFGGISGVLWAYFLLYVLDLPYFFK*.

[0011] On the other hand, the present invention relates to the use of the rice photosystem I subunit OsPsaL gene in crop breeding resistant to Tenuivirus, Reoviridae, and Fijivirus, especially in the breeding of Gramineae food crops;

[0012] In some embodiments, the Tenuiviruses include Echinochloa hoja blanca virus (EHBV), Maize stripe virus (MSpV), Rice grassy stunt virus (RGSV), Rice hoja blancavirus (RHBV), Rice stripe virus (RSV), Urochloa hojablanca virus (UHBV). The Fijiviruses include Maize Rough Dwarf Virus (MRDV), Rice black streaked dwarf virus (RBSDV), Southern rice black streaked dwarf virus (SRBSDV);

[0013] In some embodiments, the Fijivirus is preferably Rice Stripe Virus. The Fijivirus is most preferably Southern Rice Black-streaked Dwarf Virus and Rice Black-streaked Dwarf Virus.

[0014] In some embodiments, the present invention relates to an application of the rice photosystem I subunit OsPsaL gene in rice breeding that is resistant to rice stripe leaf blight and / or southern rice black-streaked dwarf virus.

[0015] In some embodiments, the gramineous food crops are preferably rice, corn, wheat, oats and barley; more preferably rice, and most preferably Nipponbare;

[0016] In some embodiments, the negative regulation of OsPsaL gene expression is achieved by the Crispr / Cas9 system; the target of the Crispr / Cas9 system is shown in SEQ ID NO:3, specifically as follows: ATCAATGGCGACCCCTTCATCGG.

[0017] In another aspect, the present invention relates to a method for preparing a transgenic plant resistant to rice stripe disease, the steps comprising:

[0018] 1) Construction of rice OsPsaL knockout vector;

[0019] 2) Rice genetic transformation, Agrobacterium transformation and callus induction culture

[0020] 3) Identification of positive transgenic plants;

[0021] In some embodiments, the rice OsPsaL knockout vector construction step further comprises:

[0022] Primers were designed on the CRISPR-GE website (http: / / skl.scau.edu.cn / ) according to the sequence of OsPsaL shown in SEQ ID NO: 1, and the gene sgRNA fragment was cloned from rice. The PCR product was recovered by gel; the target sequence was connected to the U3 promoter and gRNA scaffold respectively using the pYLsgRNA-OsU3 vector as a template. The two products obtained by PCR were mixed as templates, and the U3 promoter, target sequence and gRNA scaffold were connected together by overlapping PCR to form an sgRNA expression cassette. The gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid were mixed and digested with BsaI at 37°C for 15min. After digestion, 1.5μL 10×DNA LigaseBuffer and 35U T4 ligase were added, and 15 cycles of variable temperature cycle digestion and connection were performed, and the cycle program was 37°C 5min; 10°C 5min, 20°C 5min. Positive clones were selected and sequenced to confirm that the expression vector YL-Hu-OsPsaL had been successfully constructed. The recombinant plasmid was obtained and the quality of the plasmid was tested by 1% agarose gel electrophoresis.

[0023] In some embodiments, the steps of rice genetic transformation, Agrobacterium transformation and callus induction culture further include:

[0024] Callus induction: First, select mature rice seeds, peel off the husk, pour into a centrifuge tube, add 75% ethanol to disinfect for 3 minutes, then pour out the ethanol, rinse three times with sterile water, then add 15mL 30% sodium hypochlorite to disinfect for 20 minutes, then pour out the sodium hypochlorite and rinse 5-6 times with sterile water; use a pipette to absorb excess water, transfer the seeds to the induction medium, place in a 28℃ light incubator for 3 weeks, and use pre-sterilized tweezers to clip the grown callus into the subculture medium, and subculture in a 28℃ light incubator for 1 week;

[0025] Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101 using the following steps: 5 μL of plasmid was added to 100 μL of Agrobacterium competent medium, mixed by pipetting, added to a sterile electrode cup precooled at 4°C, and transformed by electric shock at 220 V, added to LB liquid culture medium without resistance, and cultured on a constant temperature shaker at 28°C for about 2 hours, spread on LB solid culture medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in the dark at 28°C for 2 days until single colonies appeared.

[0026] Agrobacterium transfection: Use a pipette to absorb the infection solution and wash off the Agrobacterium on the plate to obtain the Agrobacterium suspension for co-culture and rice transformation. Select a sufficient number of callus tissues, place them in a 100ml sterile triangular flask, add an appropriate amount of Agrobacterium suspension, and place them at room temperature for infection for 20 minutes, shaking them from time to time. Pour out the bacterial solution, place the callus tissue on a sterile filter paper to absorb the excess bacterial solution, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper, and culture it in the dark at 26℃ for 3 days.

[0027] Screening and culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1 ml pipette tip to transfer the callus on the co-culture medium to a sterilized conical flask, add sterile water to rinse twice, and rinse once with sterile water containing 500 μL / L carbenicillin for the third time. After the excess water is sucked out by a pipette, the callus is transferred to sterile filter paper. The water on the callus is blown dry by the wind from the clean bench. The blowing time is controlled at about 30 minutes. After the callus is dried, it is transferred to the culture medium containing hygromycin B for screening and culture. The culture conditions are 28-30℃, and the screening time is 3-4 weeks.

[0028] Rooting of seedlings: When the differentiated seedlings grow to about 2-3cm and have obvious root systems, they can be transferred to rooting medium for growth. The rooting medium should be poured into a higher bottle or tube so that the rooted seedlings have enough space to grow taller. The rooting culture conditions are 28-30℃ and sterile light culture.

[0029] In some embodiments, the step of identifying the positive transgenic plants further includes: taking young leaves of T0 and T1 generations, extracting DNA, amplifying the sgRNA targeting site with target-specific primers, performing 1% gel electrophoresis to detect the amplified product, recovering the specific and correct-sized fragments from the gel, and sequencing them. Named ospsal-ko (ospsal-1# and ospsal-2#), it proves that the transgenic rice line was successfully constructed.

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

[0031] The resistance of the overexpressing mutant ospsal-ko transgenic line to rice stripe virus disease and southern rice black-streaked dwarf virus disease was tested by artificial inoculation of RSV and SRBSDV.

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

[0037] The present invention constructs a YL-Hu-OsPsaL vector and introduces it into a rice variety (for example, Nipponbare) by Agrobacterium-mediated method to obtain two transgenic rices that can be stably inherited, which are named ospsal-1# and ospsal-2#, respectively. And the resistance analysis to rice stripe virus and southern rice black-streaked dwarf virus is detected, which helps to deepen our understanding and knowledge of rice viruses and enrich the molecular mechanism of the interaction between rice and RSV and SRBSDV. From the perspective of industrial application, the experimental results show that the OsPsaL mutant transgenic plants inoculated with RSV or SRBSDV, including the symptoms of the diseased plants and the expression of the virus at the RNA level, are significantly lower than those of the susceptible wild control NIP plants. It can be seen that the OsPsaL mutant transgenic rice can significantly enhance the resistance of rice to RSV or SRBSDV infection; the OsPsaL mutant plant has a good application value in the field of transgenics. The invention has practical guiding significance for cultivating transgenic plants resistant to rice stripe leaf blight and southern rice black-streaked dwarf virus, and also has important application prospects in the field of plant disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 :Results of OsPsaL mutation in ospsal-ko mutant transgenic rice.

[0039] Figure 2 : Symptoms of ospsal-KO mutant transgenic and control Nipponbare after RSV infection.

[0040] Figure 3 : Results of virus content detection in ospsal-KO mutant transgenic and control Nipponbare after RSV infection.

[0041] Figure 4 : Pathogenesis results of ospsal-ko mutant transgenic and control Nipponbare after SRBSDV infection. Figure 5 : Detection results of virus content in ospsal-ko mutant transgenic and control Nipponbare after SRBSDV infection.

[0042] mock: healthy rice; RSV: rice infected with rice stripe virus (RSV); SRBSDV: rice infected with southern rice black-streaked dwarf virus (SRBSDV); NIP: Nipponbare rice.

[0043] The following examples are provided to better illustrate the content of the present invention, but the content of the present invention is not limited to the examples. Those skilled in the art may make non-essential improvements and adjustments to the implementation scheme based on the above content of the invention, which still fall within the scope of protection of the present invention. DETAILED DESCRIPTION

[0044] The rice varieties used in this series of experiments are: Nipponbare

[0045] The relevant culture medium components are as follows:

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Rooting medium: 1 / 2MS (Manufacturer: Haibo Biotechnology Co., Ltd., Product No.: HB8469-6)

[0050] 39.45g / L, 0.5mg / L NAA, 50mg / L hygromycin, PH=5.8.

[0051] Example 1: Construction of rice YL-Hu-OsPsaL vector

[0052] The CRISPR / Cas9 gene editing vector construction scheme uses overlapping PCR to construct the sgRNA expression cassette. The specific steps are as follows:

[0053] 1. According to the sequence of OsPsaL shown in SEQ ID No: 1, primers were designed on the CRISPR-GE website (http: / / skl.scau.edu.cn / ), the gene sgRNA fragment was cloned from rice, and the PCR product was recovered by gel; using the pYLsgRNA-OsU3 vector as a template, the target sequence was connected to the U3 promoter and gRNA scaffold respectively. The two products obtained by PCR were mixed as a template, and the U3 promoter, target sequence and gRNA scaffold were connected together by overlapping PCR to form an sgRNA expression cassette.

[0054] The PCR primer sequences are:

[0055] SEQ ID NO:4

[0056] YL-Hu-OsPsaL-F:CAGTGGTCTCATGCAATCAATGGCGACCCCTTCATCGG;

[0057] SEQ ID NO:5

[0058] YL-Hu-OsPsaL-R:CAGTGGTCTCAAAACCCGATGAAGGGGTCGCCATTGAT

[0059] Mix the gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid, and digest them with BsaI at 37°C for 15 minutes. After digestion, add 1.5μL 10×DNALigase Buffer and 35U T4 ligase, and use variable temperature cycle digestion and ligation for 15 cycles. The cycle program is: 37°C 5min; 10°C 5min, 20°C 5min. Select positive clones, sequence, and confirm that the expression vector YL-Hu-OsPsaL has been successfully constructed. Obtain recombinant plasmids, detect plasmid quality by 1% agarose gel electrophoresis, and transform the correctly sequenced recombinant vectors into Agrobacterium GV3101.

[0060] Example 2: Rice genetic transformation

[0061] 1) Callus induction and subculture: Select newly harvested mature rice seeds, peel off the husk, pour into a 50 ml centrifuge tube, add 15 ml of 75% ethanol to disinfect for 3 minutes, pour out the ethanol, rinse three times with sterile water, pour out, add 15 ml of 30% sodium hypochlorite to disinfect for 20 minutes, pour out the sodium hypochlorite and rinse with sterile water 5-6 times. Use a pipette to suck out excess water, transfer the seeds to the induction medium, place in a 28 ° C light incubator for 3 weeks, and use pre-sterilized tweezers to clip the grown callus into the subculture medium, and subculture in a 28 ° C light incubator for 1 week.

[0062] 2) Transformation and culture of Agrobacterium: The plasmid containing the target vector was transformed into Agrobacterium tumefaciens GV3101 (manufacturer: Shanghai Weidi, product number: AC1003S) using the following steps: 5 μL of plasmid was added to 100 μL of Agrobacterium GV3101 competent medium, mixed by pipetting, added to a sterile electrode cup precooled at 4°C in advance, and transformed by electroporation at a voltage of 220 V, added to non-resistant LB liquid culture medium, and cultured on a constant temperature shaker at 28°C for about 3 hours, evenly spread on LB solid culture medium containing 50 μg / ml Kan and 50 μg / ml Rif, and cultured in the dark at 28°C for 2-3 days until single colonies appear.

[0063] 3) Agrobacterium transfection of callus: Use a pipette to absorb the infection solution and wash off the Agrobacterium on the plate to prepare the Agrobacterium suspension required for rice transformation. Select a sufficient number of callus (the callus is in good condition, bright yellow in color, round and hard in texture, and the particle diameter is about 3mm), put it into a 100ml sterile conical flask, add an appropriate amount of Agrobacterium suspension (make sure there is enough bacterial liquid in contact with the material), and place it at room temperature for transfection for 20 minutes, shaking it from time to time. Pour out the bacterial liquid, place the callus on sterile filter paper to absorb the excess bacterial liquid, and then transfer it to a solid co-culture medium covered with a layer of sterile filter paper, and culture it in the dark at 26℃ for 3 days.

[0064] 4) Screening culture: After 3 days of co-culture, the callus tissue needs to be cleaned. Use a 1ml pipette tip to transfer the callus on the co-culture medium to a sterilized Erlenmeyer flask and rinse twice with sterile water. Rinse once with sterile water containing 500ul / L carbenicillin for the third time. After the excess water is sucked out with a pipette, the callus is transferred to sterile filter paper and the water on the callus is blown dry with the wind from the clean bench. The blowing time is controlled to about 30 minutes. After the callus is dried, it is transferred to the screening culture medium for screening culture. The culture conditions are 28-30℃ and dark culture; the screening time is 3-4 weeks.

[0065] 5) Rooting of seedlings: When the seedlings grow to about 2-3 cm and have obvious roots, they are transferred to rooting medium for further growth. The rooting medium should be in a higher bottle or tube so that the seedlings have enough space to grow upwards. The best conditions for rooting are sterile light culture at 28-30 degrees Celsius. Positive transgenic plants ospsal-1# and ospsal-2# were obtained.

[0066] Example 3: Identification of positive transgenic plants

[0067] Take the young leaves of T0 and T1 generations, extract DNA by CTAB method, amplify the sgRNA targeting site with target-specific primers, detect the amplified product by 1% gel electrophoresis, and recover the specific and correct-sized fragments for sequencing. The sequencing primers are as follows:

[0068] SEQ ID NO:6

[0069] OsPsaL-F:GAATTAGAAGTGATTGATGGGAC

[0070] SEQ ID NO:7

[0071] OsPsaL-R:AAAGATCTACTTGAAGAAGTACG

[0072] The experimental results are as follows Figure 1As shown, the sequencing results showed that ospsal-1# inserted a base T at the target site, OsPsaL mutated 105 amino acids, while ospsal-2# lacked seven bases at the target site, causing OsPsaL to terminate prematurely. This proved that the transgenic rice line was successfully constructed.

[0073] Example 4: Artificial inoculation of RSV

[0074] (1) After soaking and germinating the seeds of ospsal-ko transgenic plants and control Nipponbare rice materials for 2-3 days, the seeds were sown in 1 L beakers after they turned white, with about 30 seedlings per beaker, and three biological replicates. The plants were cultured at 30°C with 16 h of light and 8 h of darkness.

[0075] (2) Virus inoculation experiments were conducted using RSV-carrying and healthy gray leafhoppers at the 3rd or 4th instar stage. RSV-carrying and virus-free gray leafhoppers were inoculated on rice plants at the 3rd or 4th leaf stage at a ratio of 2 to 3 per seedling. After feeding for 3 days, all insects were swept out.

[0076] (3) After 30 days, the symptoms of diseased rice were observed and the virus status of rice was determined by qRT-PCR.

[0077] Example 5: Resistance analysis of rice after RSV inoculation

[0078] 20 days after transplanting rice, RSV-infected rice showed mosaic symptoms. Compared with the control Nipponbare NIP, the two ospsal-ko mutant transgenic lines (ospsal-1# and ospsal-2#) showed mild mosaic symptoms, while the control NIP showed obvious mosaic symptoms, such as Figure 2 The diseased rice plants were mixed and sampled, with 3 biological replicates in each group, and the expression of the RSV CP gene was detected by qRT-PCR, as shown in Figure 3 As shown, the expression level of CP gene in transgenic lines was significantly lower than that in controls. The above results indicate that the resistance of transgenic plants to RSV infection is affected by the OsPsaL gene, and the mutation of OsPsaL in rice can significantly enhance the resistance of rice to RSV infection.

[0079] The sequences of the quantitative primers are as follows:

[0080] qRSV-CP-F AGGCAATCAATGACATCTCC; SEQ ID NO:8

[0081] qRSV-CP-R ATCTCTCACAAAGCCAGTGC; SEQ ID NO:9

[0082] In summary, the experimental results show that compared with the control NIP, the ospsal-ko mutant transgenic rice plants can significantly enhance the resistance of rice to RSV infection (including phenotypic symptoms and viral load of diseased plants). Therefore, the present invention successfully obtained RSV infection-resistant transgenic rice through gene editing, which is of great significance to agricultural development and national food security, and achieved unexpected technical effects.

[0083] Example 6: Artificial inoculation of SRBSDV

[0084] (1) After soaking and germinating the seeds of ospsal-ko transgenic plants and control Nipponbare rice materials for 2-3 days, the seeds were sown in 1 L beakers after they turned white, with about 30 seedlings per beaker, and three biological replicates. The beakers were cultured at 30°C and 16 h light and 8 h dark conditions.

[0085] (2) The newly hatched 1-2-year-old non-virus-infected white-backed planthoppers were infected with SRBSDV for 3-5 days on rice seedlings infected with SRBSDV. After a 10-day circulation period, white-backed planthoppers that can be used to spread SRBSDV were obtained. The virus inoculation experiment was carried out with white-backed planthoppers carrying SRBSDV and healthy white-backed planthoppers of the same age. The virus-infected / non-virus-infected white-backed planthoppers were inoculated on rice seedlings at the three- to four-leaf stage (about 15 days) at a ratio of 3 per seedling. After feeding for 3 days, all the insects were swept out.

[0086] (3) After 30 days, the symptoms of diseased rice were observed and the virus status of rice was determined by qRT-PCR.

[0087] Example 7: Resistance analysis of rice after inoculation with SRBSDV

[0088] 30 days after transplanting rice, it was found that the rice infected by SRBSDV showed dwarfing symptoms. Compared with the control Nipponbare NIP, the two ospsal-ko mutant transgenic lines (ospsal-1# and ospsal-2#) showed mild dwarfing symptoms, while the control NIP showed obvious dwarfing symptoms, such as Figure 4 The diseased rice plants were mixed and sampled, with 3 biological replicates in each group, and the expression levels of SRBSDV S2, S4, and S6 genes were detected by qRT-PCR. Figure 5 As shown, the expression levels of S2, S4, and S6 genes of the transgenic strains were significantly lower than those of the control. The above results indicate that the resistance of transgenic plants to SRBSDV infection is affected by the OsINT1 gene, and the mutation of OsPsaL in rice can significantly enhance the resistance of rice to SRBSDV infection.

[0089] The sequences of the quantitative primers are as follows:

[0090] qSRB-S2-F:CATCGACCAAGTTCAACCCG; SEQ ID NO:10

[0091] qSRB-S2-R:AAGAAGTCTGCGGGTGAAGA; SEQ ID NO:11

[0092] qSRB-S4-F:AAAGTGAACCCGTTGCTGAC; SEQ ID NO:12

[0093] qSRB-S4-R:TGCAACGCTAGATCCTATGC; SEQ ID NO:13

[0094] qSRB-S6-F:ATCTGCTTTCCCCTTCCGA; SEQ ID NO:14

[0095] qSRB-S6-R:GATTCCGCGTTTGAAGAGTCA; SEQ ID NO:15

[0096] In summary, the experimental results show that compared with the control NIP, the ospsal-ko mutant transgenic rice plants can significantly enhance the resistance of rice to SRBSDV infection (including phenotypic symptoms and viral load of diseased plants). Therefore, the present invention successfully obtained SRBSDV infection-resistant transgenic rice through gene editing, which is of great significance to agricultural development and national food security, and achieved unexpected technical effects.

Claims

1. Application of the rice photosystem I subunit OsPsaL gene in rice breeding for resistance to rice stripe virus and southern rice black-streaked dwarf virus, wherein the gene sequence is shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the protein sequence encoding the OsPsaL gene is shown in SEQ ID NO:

2.

3. The use according to any one of claims 1 to 2, wherein the resistance of rice to rice stripe virus (RSV) and southern rice blackstreaked dwarf virus (SRBSDV) is increased by negatively regulating the expression of the OsPsaL gene.

4. The use according to any one of claim 3, wherein the negative regulation of the expression of the OsPsaL gene is achieved by the Crispr / Cas9 system; the target of the Crispr / Cas9 system is shown in SEQ ID NO:

3.

5. A method for preparing transgenic rice resistant to rice stripe virus (RSV) and southern rice black streaked dwarf virus (SRBSDV), comprising the steps of: 1) Construction of rice OsPsaL gene knockout vector; 2) Rice genetic transformation, Agrobacterium transformation and callus induction culture; 3) Identification of positive transgenic plants.

6. The method according to claim 5, wherein the construction of the rice OsPsaL gene knockout vector comprises the following steps: Primers were designed according to the sequence of OsPsaL shown in SEQ ID NO: 1, and the gene sgRNA fragment was cloned from rice, and the PCR product was recovered from gel; the target sequence was connected to the U3 promoter and gRNA scaffold respectively using the pYLsgRNA-OsU3 vector as a template; the two products obtained by PCR were mixed as templates, and the U3 promoter, target sequence and gRNA scaffold were connected together by overlapping PCR to form an sgRNA expression cassette; the gRNA expression cassette product and the uncut pYLCRISPR / Cas9Pubi-H plasmid were mixed, and they were digested with BsaI at 37°C for 15 min; after digestion, 1.5 μL 10×DNA Ligase Buffer and 35U T4 ligase were added, and 15 cycles of variable temperature cycle digestion and connection were performed, and the cycle program was: 37°C 5 min; 10°C 5 min, 20°C 5 min; positive clones were selected and sequenced to confirm that the rice OsPsaL gene knockout expression vector YL-Hu-OsPsaL had been successfully constructed.

7. The method according to claim 6, wherein the primer sequences are as follows: YL-Hu-OsPsaL-F: CAGTGGTCTCATGCAATCAATGGCGACCCCTTCATCGG; YL-Hu-OsPsaL-R: CAGTGGTCTCAAAACCCGATGAAGGGGTCGCCATTGAT.

8. A method for improving rice resistance to rice stripe virus and southern rice black-streaked dwarf virus, using CRISPR / Cas9 technology to target and edit the rice photosystem I subunit OsPsaL gene, wherein the nucleotide sequence of the OsPsaL gene is shown in SEQ ID NO.

1.

9. An sgRNA, whose target sequence binding region is shown in SEQ ID NO: 3.

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