Application of rice transcription factor OsLSD5 in rice antiviral

Abstract

The present application relates to a kind of rice transcription factor OsLSD5 and its application in anti-rice stripe virus, by the overexpression OsLSD5 vector after being transferred in plant rice, obtain the stable genetic OsLSD5-ox transgenic plant.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant disease prevention and control and transgenic technology, and mainly relates to a rice transcription factor OsLSD5 and application thereof in rice viral disease resistance. BACKGROUND

[0002] Rice stripe virus (RSV) is currently considered to be one of the most important viral pathogens of rice in East Asia, and is a typical representative of the Tenuivirus genus. Rice plants infected with RSV usually exhibit symptoms such as wilting, discontinuous yellow stripes, necrosis of newly emerged leaves, and growth retardation. The virus is mainly transmitted by Laodelphax striatellus and can be transmitted to offspring through the ovaries of insects. With the development of biotechnology, researchers have conducted a large number of studies on the genomics, proteomics of RSV, and the interaction between different viral proteins encoded by RSV and host factors, and found that the RSV genome is composed of four negative single-stranded RNAs, which are named RNA1, RNA2, RNA3 and RNA4 according to their size, and encode 7 proteins.

[0003] Lesion simulating disease (LSD) proteins are a specific zinc finger protein subfamily, belong to C2C2 type transcription factors, contain multiple conserved zinc finger domains CxxCRxxLMYxxGASxVxCxxC, and this zinc finger sequence has homology with GATA type transcription factors, exists in plants, algae and protozoa, but does not exist in animals. PAD4 (Enhanced disease susceptibility 1) can encode a pathogen-induced protein that plays a role upstream of salicylic acid (SA). EDS1 (Enhanced disease susceptibility 1) is an important regulator of innate immunity, and participates in R gene signal transduction together with PAD4. Studies have shown that the PCD symptoms exhibited by lsd1 Arabidopsis mutants depend on EDS1 and PAD4. LSD1, EDS1 and PAD4 form a specific central core responsible for triggering cell death dependent on salicylic acid, ethylene and reactive oxygen species and adaptive responses to adverse environments.

[0004] LSDs play an important role in plant growth and development, abiotic and biotic stress response. Rice LSD1 can interact with transcription factor OsbZIP58, promote the binding of OsbZIP58 to the promoter of gibberellin synthesis related gene KO2, up-regulate the expression of OsKO2, thereby activating gibberellin synthesis, and gibberellin can activate amylase expression and promote seed germination. On the other hand, OsLSD1 can indirectly down-regulate the expression of OssodCc1, reduce the activity of superoxide dismutase SOD, and increase the accumulation of reactive oxygen species, accelerate the PCD of the aleurone layer, and promote seed germination. Overexpression of rice gene OsLOL5 significantly up-regulates rice oxidative stress related genes OsAPX2, OsCAT, OsCu / Zn-SOD and OsRGRC2, and increases the resistance of rice to salt stress. Phakopsora pachyrhizi is a pathogenic strain of Asian rust. When the rust fungus infects soybeans, the expression level of soybean LSD increases rapidly. Silencing of wheat gene TaLSD1 can promote hypersensitive cell death in wheat and increase the resistance of wheat to stripe rust. Overexpression of rice gene OsLOL2 can enhance the resistance of rice to rice bacterial leaf blight. Cassava LSD3 can interact with deacetylase SRT1, inhibit its deacetylation modification on histone H3K9Ac, increase the expression level of H3K9Ac, up-regulate the expression of pattern recognition receptor genes SOBIR1 (Suppressor of BIR 1) and FLS2 (Flagellin-sensitive 2), and resist Xanthomonas phaseoli pv. manihotis infection in cassava. These studies show that LSD proteins play an important role in plant resistance to pathogen infection. However, there is no report on the relationship between rice virus and LSD protein. SUMMARY

[0005] Based on the defects of the prior art, the present application relates to a rice LSD gene and its application in resisting rice virus.

[0006] On the one hand, a rice transcription factor OsLSD5 has the following nucleotide and protein sequences:

[0007] The nucleotide sequence of OsLSD5 is shown as SEQ ID NO: 1:

[0008] ATGCAGGACCAGCTGATCTGCAGCGGCTGCAGGCGCGTCGTCCAGTACAGGAGAGGGGTCGCCGGCGTCTGCTGCCCGGGCTGCAACACGCTCACCGCCGTCAACCCGTCAGCGGTGGCCGACATGTCGGAGCTCATCTGCAGCGGCTGCCCCACGCTGCTGTTCTACAACCGCGGCGCCTCCAACATCCGCTGCCCCAGCTGCAACAGGCTCAACTCCACCAGATCAGCCAACCAGATTGCACACCTGACATGCGGGCAGTGCCGGACGACTCTGATGCACCCACCTGGAGCCTCAACTGTGCAGTGTGCAACCTGCAGATATGTTAACCATGTCAGGGATGCTCGGCCTCAAACTGTCCTTGTAGAGAATCCTAAGACACTGGATGATAAGGGCAAGCTGGTGAGCAATGTGGTTGTTGGTGTCACCTCATGGAAAAGATGA

[0009] The amino acid sequence of OsLSD5 is shown as SEQ ID NO: 2:

[0010] MQDQLICSGCRRVVQYRRGVAGVCCPGCNTLTAVNPSAVADMSELICSGCPTLLFYNRGASNIRCPSCNRL

[0011] NSTRSANQIAHLTCGQCRTTLMHPPGASTVQCATCRYVNHVRDARPQTVLVENPKTLDDKGKLVSNVVVGVTSWKR*

[0012] In some embodiments, the biological material related to the protein encoded by the rice gene OsLSD5 provided by the present application is any one of the following A1) to A12):

[0013] A1) a nucleic acid molecule encoding the protein of the OsLSD5 gene;

[0014] A2) an expression cassette containing the nucleic acid molecule of A1);

[0015] A3) a recombinant vector containing the nucleic acid molecule of A1);

[0016] A4) a recombinant vector containing the expression cassette of A2);

[0017] A5) a recombinant microorganism comprising the nucleic acid molecule of A1);

[0018] A6) a recombinant microorganism comprising the expression cassette of A2);

[0019] A7) a recombinant microorganism comprising the recombinant vector of A3);

[0020] A8) a recombinant microorganism comprising the recombinant vector of A4);

[0021] A9) a transgenic plant cell line comprising the nucleic acid molecule of A1);

[0022] A10) a transgenic plant cell line comprising the expression cassette of A2);

[0023] A11) a transgenic plant cell line comprising the recombinant vector of A3);

[0024] A12) a transgenic plant cell line comprising the recombinant vector of A4).

[0025] In another aspect, the present application relates to the use of the rice transcription factor OsLSD5 in breeding against Tenuivirus in food crops of the family Poaceae;

[0026] In some embodiments, the Tenuivirus comprises Rice stripe virus (RSV), Maize stripe virus (MSpV), preferably Rice stripe virus (RSV);

[0027] In some embodiments, the food crops of the family Poaceae preferably rice, corn, wheat, oat and barley; more preferably rice, most preferably Zhonghua 11;

[0028] In another aspect, the present application relates to a method for preparing a transgenic plant against Rice stripe virus and / or against Southern rice black-streaked dwarf virus, the steps of which comprise:

[0029] (1) cloning of the rice OsLSD5 gene;

[0030] (2) construction of the overexpression vector;

[0031] (3) Agrobacterium transformation and callus induction culture;

[0032] (4) positive identification of the transgenic plant;

[0033] In some embodiments, the cloning of the rice OsLSD5 gene comprises the following steps:

[0034] 1) Design primers OsLSD5-F and OsLSD5-R according to the open reading frame (ORF) of OsLSD5

[0035] ATGCAGGACCAGCTGATCTG (SEQ ID NO: 3);

[0036] TCATCTTTTCCATGAGGTGACACC (SEQ ID NO: 4);

[0037] 2) Perform PCR reaction, recover the PCR product, ligate pMD18-T vector, pick single clone, send for testing, and obtain correct pMD18-T-OsLSD5 recombinant plasmid.

[0038] In some embodiments, the construction of the overexpression vector comprises the following steps:

[0039] 1) Design recombinant primers with GTCACC and CCGTCG enzyme cutting sites, use correct pMD18-T-OsLSD5 recombinant plasmid as template to amplify OsLSD5 gene, and recover the PCR product; then use ApaI enzyme to double cut the pCV1300 vector, and recover the cut vector;

[0040] 2) Ligate the above PCR product and vector, transform E. coli DH5a, pick positive clones, perform sequencing, and confirm that the pCV1300-OsLSD5 overexpression vector has been successfully constructed;

[0041] The recombinant primer sequences are as follows:

[0042] pCV-OsLSD5-F CGACGACAAGACCGTCACCATGCAGGACCAGCTGATCTG (SEQ ID NO: 5);

[0043] pCV-OsLSD5-R GAGGAGAAGAGCCGTCGTCTTTTCCATGAGGTGACACC (SEQ ID NO: 6);

[0044] In some embodiments, the Agrobacterium transformation and callus induction culture step comprises:

[0045] (1) Take an appropriate amount of the constructed overexpression vector plasmid and add it to Agrobacterium competent cells. Mix well and add it to an electrode cup pre-cooled at 4℃. Transform with a voltage of 2200V. Add 800μL of LB liquid medium without resistance. Incubate at 28℃ in a shaker for 2-3h. Centrifuge at 5000rpm for 1min. Discard the supernatant. Spread the remaining precipitate on solid medium containing 50μg / ml Kan and 50μg / ml Rif resistance. Incubate at 28℃ for 3d. Dehull the rice seeds to be transformed. Soak in 75% alcohol for 5min. Rinse twice with sterile water. Soak in 30% sodium hypochlorite solution for 30min. Soak in sterile water for 30min. Place the seeds in the induction medium with tweezers. Incubate at 28℃ in a light incubator for 3-4 weeks. Transfer the callus tissue that grows to the subculture medium with pre-sterilized tweezers. Subculture at 28℃ in a light incubator for 1 week.

[0046] (2) Select a single colony of Agrobacterium GV3101 and inoculate it into LB liquid medium, and culture the bacterial solution to the OD concentration. 600 = Approximately 0.6; Immerse the induced callus in Agrobacterium suspension culture for 5 minutes; Remove the callus and place it in a symbiotic culture medium, and culture in a 26℃ light incubator for 2-3 days; Then transfer the callus to a culture medium containing hygromycin and grow for 30-45 days; Finally, place the screened callus in a rooting medium and culture under light until the callus roots, and continue culture for 2 weeks to obtain transgenic plants.

[0047] In some implementations, the positive seedling identification step of the transgenic plant includes:

[0048] Total RNA was extracted from transgenic plants that had undergone hygromycin screening and quantitatively reverse transcribed into cDNA, with the OsUBQ5 gene of rice used as an internal control; the quantitative primers for OsLSD5 are shown in SEQ ID NO:7-10.

[0049] qRT-OsLSD5-F:GACATGTCGGAGCTCATCTG (SEQ ID NO:7);

[0050] qRT-OsLSD5-R: CAATCTGGTTGGCTGATCTG (SEQ ID NO: 8);

[0051] OsUBQ5-F:ACCACTTCGACCGCCACTACT(SEQ ID NO:9);

[0052] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10);

[0053] In some embodiments, the induction medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, PH = 5.8.

[0054] 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.

[0055] The co-culture medium comprises: N6 medium 24.1 g / L, 2 mg / L 2,4-D, 200 μmol / L acetosyringone, PH = 5.2.

[0056] The rooting medium comprises: 1 / 2MS 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH = 5.8.

[0057] In another aspect, the present application also relates to a method for detecting the resistance of rice to rice stripe virus, comprising the following steps:

[0058] 1) Extract total RNA from the rice to be tested and the control Zhonghua 11 rice by using TRIzol method, and reverse transcribe the RNA into cDNA; detect the relative expression amount of OsLSD5 gene by using quantitative primers, wherein the quantitative primers are as follows:

[0059] qRT-OsLSD5-F: GACATGTCGGAGCTCATCTG (SEQ ID NO: 7);

[0060] qRT-OsLSD5-R: CAATCTGGTTGGCTGATCTG (SEQ ID NO: 8);

[0061] OsUBQ5-F: ACCACTTCGACCGCCACTACT (SEQ ID NO: 9);

[0062] OsUBQ5-R: ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10);

[0063] According to the detection result of the gene expression amount, it is determined whether the rice plant to be tested is a resistant rice, for example, if the relative expression amount of OsLSD5 gene of the rice plant to be tested is significantly higher than that of the control, then it is a resistant rice.

[0064] After the full-length OsLSD5 gene is transformed into rice by using plant transgenic technology, a transgenic line with high expression amount is screened. The experiment shows that after the RSV infects the rice, the overexpression of OsLSD5 transgene significantly increases the resistance to virus infection compared with the wild type control. OsLSD5 plays a positive regulation role in the process of rice resistance to virus.

[0065] The application mainly adopts the following scheme to realize the above application purposes:

[0066] The application takes rice transcription factor OsLSD5 as the object, constructs an OsLSD5 recombinant expression vector pCV1300-OsLSD5, and transmits the vector into ZH11 embryos by using an agrobacterium GV3101 mediated genetic transformation method, and then is placed into a symbiotic culture medium to induce the formation of callus. After obtaining the transgenic rice seedlings, the expression amount of the OsLSD5 gene in the rice is detected by using a real-time fluorescent quantitative PCR (qRT-PCR) technology, and after several generations of screening and identification, the T3 generation of the stable genetically pure transgenic rice is obtained. The OsLSD5 transgenic rice is subjected to resistance evaluation by artificial inoculation of RSV. The results show that the symptoms of the transgenic rice overexpressing OsLSD5 after infection of RSV are lighter than those of the wild type, and the virus content in the body is also significantly lower than that of the wild type rice. Therefore, it is indicated that the OsLSD5 overexpression transgenic rice can significantly enhance the resistance of the rice to RSV. The research results not only show that the OsLSD5 affects the infection efficiency of RSV, but also lay a foundation for the research on the interaction between the rice and the virus.

[0067] Compared with the prior art, the application has the following advantages and beneficial effects:

[0068] (1) The application overexpresses OsLSD5 in ZH11 rice, obtains the stable genetically overexpressed OsLSD5 transgenic rice, and analyzes the resistance of the transgenic rice to rice stripe disease, which helps to deepen our understanding and recognition of the virus and host factors of the rice, and enriches the molecular mechanism of the interaction between the virus and the rice.

[0069] (2) The application studies the mechanism of the role of OsLSD5 in virus infection, finds the interaction relationship between the plant and the virus, and lays a foundation for further research on the disease resistance theory of the rice. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 : Experimental results of the relative expression amount of OsLSD5 in the OsLSD5 overexpression transgenic rice.

[0071] Figure 2 : Comparison results of the disease symptoms of the OsLSD5 overexpression transgenic rice and the control wild type after RSV infection.

[0072] Figure 3 : Experimental results of the virus content of the OsLSD5 overexpression transgenic rice and the control wild type after RSV infection. DETAILED DESCRIPTION

[0073] The rice variety selected in the series of experiments is ZH11.

[0074] The relevant culture medium components are as follows:

[0075] Induction medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601)

[0076] 24.1 g / L, 2.5 mg / L 2,4-D, PH = 5.8.

[0077] 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 cefotaxime, PH = 5.8.

[0078] Co-culture medium: N6 medium (manufacturer: Haibo Biotechnology Co., Ltd., product number: HBZ0601) 24.1 g / L, 2.5 mg / L 2,4-D, 200 μmol / L acetosyringone, PH = 5.8.

[0079] Rooting medium: 1 / 2MS (manufacturer: Haibo Biotechnology Co., Ltd., product number: HB8469-6)

[0080] 39.45 g / L, 0.5 mg / L NAA, 50 mg / L hygromycin, PH = 5.8.

[0081] Example 1: Construction of rice OsLSD5 overexpression vector

[0082] (1) Cloning of rice OsLSD5 gene

[0083] Primers OsLSD5-F and OsLSD5-R were designed according to the open reading frame (ORF) of OsLSD5, and the primer sequences are as follows:

[0084] Table 1: Primer sequences involved in gene cloning

[0085] OsLSD5-F ATGCAGGACCAGCTGATCTG (SEQ ID NO: 3); OsLSD5-R TCATCTTTTCCATGAGGTGACACC (SEQ ID NO: 4);

[0086] PCR amplification system: total volume 50 μL, including 2x PCR Buffer for KOD FX Neo 25 μL, 1.5 μL of each of the upper and lower primers (10 μM), 5 μL of dNTP Mix (2.5 mM), 1 μL of cDNA template, 1 μL of KOD FX Neo, and 15 μL of ddH2O.

[0087] PCR amplification program: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 3 min, 40 cycles; 72°C final extension for 10 min.

[0088] The PCR product was recovered, ligated with pMD18-T vector, and single clones were selected and sent for testing. The correct pMD18-T-OsLSD5 recombinant plasmid was obtained, and was sent to Hangzhou Yikang Biotechnology Co., Ltd. for sequencing.

[0089] (2) Construction of overexpression vector

[0090] A primer with BamHI and SacI restriction sites was designed to amplify OsLSD5, and the template was the correctly sequenced pMD18-T-OsLSD5 plasmid. The pCV1300 vector was double-digested with BamHI and SacI for the construction of the OsLSD5 gene binary expression vector PCV1300, and the primer sequence was as follows:

[0091] Table 2: Primer sequences involved in the construction of the overexpression vector

[0092]

[0093] PCR amplification system: The reaction system (50 μL) was as follows: 2x PCR KOD FX Neo Buffer 25 μL, 2 mM dNTPs, KOD FX Neo 1 μL, upstream primer 1.5 μL, downstream primer 1.5 μL, cDNA 1 μL, ddH2O 15 μL, and the total volume was 50 μL.

[0094] PCR program: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 2 min, 35 cycles; and 72°C final extension for 10 min.

[0095] After the pCV1300 vector was digested, the PCR product was recovered, and the above PCR product was ligated with the vector, respectively. Positive clones were selected and sequenced to confirm that the pCV1300-OsLSD5 expression vector had been successfully constructed.

[0096] Example 2: Genetic transformation of rice

[0097] (1) Agrobacterium transformation and callus induction culture: The constructed recombinant vector plasmid was transformed into Agrobacterium GV3101. The specific steps included: taking 2-3 μL of plasmid and adding it to 100 μL of competent cells, mixing well, aspirating into an electrode cup pre-cooled at 4℃, transforming with a voltage of 2200V, adding 800 μL of antibiotic-free LB liquid medium, and incubating at 28℃ and 200 rpm for 2-3 h on a shaker, centrifuging at 5000 rpm for 1 min, discarding part of the supernatant, and spreading the remaining precipitate on a plate containing 50 μg / ml Kan and 50 μg / ml Rif solid medium, and incubating at 28℃ for 3 days. Mature rice seeds were dehulled, soaked in 75% alcohol for 5 min, rinsed several times with ddH2O, soaked in 30% sodium hypochlorite solution for 30 min, and soaked in ddH2O for 30 min. Use tweezers to place the seeds into the induction medium and incubate them in a 28°C light incubator for 3 weeks. Then, use pre-sterilized tweezers to transfer the callus tissue into the subculture medium and subculture it in a 28°C light incubator for 1 week.

[0098] (2) Agrobacterium-mediated callus transfection and screening and culture of resistant callus: Single colonies of GV3101 were picked and inoculated into LB liquid medium and cultured until OD. 600 = Approximately 0.6. The induced callus tissue was immersed in Agrobacterium suspension culture for 5 min, and then the immersed callus tissue was transferred to culture medium and cultured in a 26℃ light incubator for 2-3 days. Then, the callus tissue was placed on a culture medium containing hygromycin and grown for 30-45 days. The screened callus tissue was transferred to rooting medium and cultured at 26℃ light for 2 weeks to produce green plants with roots. The OsLSD5-ox transgenic lines OsLSD5-6# and OsLSD5-8#, which overexpress the OsLSD5 gene, were obtained.

[0099] Example 3: Positive identification of transgenic plants

[0100] Total RNA was extracted from the transgenic plants to be tested and reverse transcribed into cDNA, with the OsUBQ5 gene of rice used as an internal control. Quantitative primers are shown in SEQ ID No:7-10. The relative expression levels of the OsLSD5-ox transgenic lines OsLSD5-#6 and OsLSD5-#8, which overexpress the OsLSD5 gene, were significantly higher than the control. Figure 1 ).

[0101] Table 3: Primers involved in positive identification of transgenic plants

[0102] qRT-OsLSD5-F GACATGTCGGAGCTCATCTG (SEQ ID NO: 7); qRT-OsLSD5-R CAATCTGGTTGGCTGATCTG (SEQ ID NO: 8); OsUBQ5-F ACCACTTCGACCGCCACTACT (SEQ ID NO: 9); OsUBQ5-R ACGCCTAAGCCTGCTGGTT (SEQ ID NO: 10);

[0103] Example 4: Artificial inoculation of RSV

[0104] OsLSD5 transgenic rice seeds and control rice seeds were soaked and cultured in a 37℃ incubator for 2-3 days. After the seeds sprouted white, they were sown in 1L glass beakers with 30-35 seedlings per beaker, with 3 biological replicates. The beakers were placed in a 25℃ artificial climate chamber and cultured under 16h light and 8h dark conditions. 1-2 instar non-virulent planthoppers were transferred to RSV-infected rice seedlings to acquire the virus. After 3-5 days, the planthoppers were transferred to healthy rice seedlings using a vacuum pump to complete the cycle (10-12 days).

[0105] The virus-carrying rate of the insects was detected, and the number of insects inoculated per seedling was calculated based on the virus-carrying rate. Virus-carrying / non-virus-carrying planthoppers were inoculated onto rice seedlings at the three-to-four-leaf stage (approximately 15 days old). After 3 days, all insects were removed, and the seedlings were placed in a 30℃ greenhouse for further growth. After 30 days, the rice disease symptoms were observed, and the incidence rate was statistically analyzed to determine the disease situation. Two OsLSD5-ox transgenic lines (#6 and #8) showed significant resistance to RSV, such as... Figure 2 As shown. qRT-PCR was used to further detect the virus content, and the results showed that the virus content in the infected transgenic rice lines was significantly lower than that in the wild type, such as... Figure 3 As shown.

[0106] The quantitative primer sequences are as follows:

[0107] qRSV-CP-F AGGCAATCAATGACATCTCC; SEQ ID NO:11

[0108] qRSV-CP-R ATCTCTCACAAAGCCAGTGC; SEQ ID NO:12

[0109] It should be understood that the specific examples and solutions described in this invention are given as examples for illustrative purposes only and are not intended to limit the invention. All further modifications or variations, including those within the spirit and scope of the invention, are considered to be covered within the scope of the invention.

Claims

1. The application of a rice transcription factor OsLSD5 in rice breeding resistant to rice stripe virus (RSV); the nucleotide sequence of the OsLSD5 gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, wherein the amino acid sequence of the OsLSD5 gene is shown in SEQ ID NO:

2.

3. A method for preparing resistant transgenic rice, comprising the following steps: transferring wild-type rice into an OsLSD5 overexpression vector to obtain resistant transgenic rice, wherein the resistant transgenic rice, compared with wild-type rice, has resistance to rice stripe virus (RSV), and the nucleotide sequence of the OsLSD5 gene is shown in SEQ ID NO:1.

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