Tobacco antiviral protein gene NbSRC2 and application of tobacco antiviral protein gene NbSRC2 in inhibition of plant virus infection of tobacco

Abstract

The invention discloses a tobacco antiviral protein gene NbSRC2 and application of the tobacco antiviral protein gene NbSRC2 in inhibiting tobacco infected by plant viruses, and belongs to the technical field of biology and molecular biology. The amino acid sequence of the tobacco antiviral protein gene NbSRC2 is as shown in SEQ ID NO: 2, and the nucleotide sequence is as shown in SEQ ID NO: 1. The NbSRC2 is silenced through a VIGS technology, and the virus coat protein CP mRNA, the virus coat protein CP and the virus infection symptom difference show that the silent NbSRC2 up-regulates the sensitivity of nicotiana benthamiana to TMV infection and promotes virus infection. The TMV infection is inhibited by overexpression of the NbSRC2. The NbSRC2 can inhibit the infection of plant viruses, is a potential antiviral gene on tobacco, can be used as a potential antiviral protein, and further enriches the scope of antiviral genes.

Description

Technical Field

[0001] The invention belongs to the technical field of biology and molecular biology, and specifically relates to a tobacco antiviral protein gene NbSRC2 and an application thereof in inhibiting plant viruses from infecting tobacco. Background Art

[0002] Tobacco viral diseases are one of the main diseases in tobacco-growing areas around the world, causing huge economic losses every year. There are 16 viruses that cause tobacco viral diseases, of which four are widely distributed and seriously harmful: tobacco common mosaic virus (TMV), tobacco cucumber mosaic virus (CMV), tobacco etch virus (TEV), potato virus Y (PVY), followed by tobacco ringspot virus (TRsv), potato virus X (PVX), tobacco necrosis virus (TNv), tobacco streak virus (TSV), etc. Among them, TMV is the most serious. TMV can occur during the growth of tobacco plants from the seedling stage to the entire growth and development period of the field, mainly causing the thickness of tobacco leaves to be uneven or even deformed, wrinkled and yellow-green mottled. In severe cases, the flower organs can be deformed, the fruits are small and wrinkled, the seeds cannot germinate, and the color, taste and quality of flue-cured tobacco will be reduced to varying degrees. It is estimated that due to the harm of viral diseases, the world causes economic losses of more than 100 million US dollars each year due to tobacco mosaic virus disease alone.

[0003] Once tobacco virus disease occurs, few chemical methods can be used to control it in time. For the prevention and control of virus disease, we should strengthen the prediction and forecast of pests and diseases, grasp the dynamics of virus disease occurrence in time, and take targeted prevention and control measures. Generally, we adopt a strategy of prevention first and comprehensive prevention and control.

[0004] For more than half a century, chemical pesticides have always occupied a very important position in agricultural production. However, the irrational use of chemical pesticides and their high toxicity and high residue characteristics not only pose a great threat to the safety of humans and animals, but also seriously damage the ecological balance. As the resistance of pests and diseases increases year by year, the amount and frequency of pesticide use also increase accordingly, while at the same time causing more serious vicious cycles of environmental pollution and lethality to non-target organisms. Biological pesticides are low in toxicity, reproduce quickly, do not harm natural enemies, and the objects of control are not easy to develop resistance, so they are called "harmless pesticides." Therefore, the development of antiviral genes is of great significance for the development of biological pesticides for the control of plant viruses. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to provide a tobacco antiviral protein gene NbSRC2 and its application in inhibiting plant viruses from infecting tobacco.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A tobacco antiviral protein gene NbSRC2, the amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0008] Based on the above scheme, the nucleic acid sequence of the gene is shown in SEQ ID NO:1.

[0009] The application of the tobacco antiviral protein gene NbSRC2 in inhibiting plant viruses from infecting tobacco.

[0010] Based on the above scheme, the plant virus is at least one of tobacco common mosaic virus (TMV), tobacco cucumber mosaic virus (CMV) and potato virus Y (PVY).

[0011] A method for inhibiting plant viruses from infecting tobacco comprises constructing a gene sequence of NbSRC2 into an expression vector, transforming tobacco, and overexpressing the gene in tobacco, thereby inhibiting plant viruses from infecting tobacco; the nucleic acid sequence of the NbSRC2 gene is shown in SEQ ID NO: 1.

[0012] Based on the above scheme, the expression vector is any one of a prokaryotic expression vector, a eukaryotic expression vector and a viral expression vector.

[0013] Based on the above scheme, the plant virus is at least one of tobacco common mosaic virus (TMV), tobacco cucumber mosaic virus (CMV) and potato virus Y (PVY).

[0014] Based on the above scheme, the plant virus is tobacco common mosaic virus.

[0015] A recombinant vector, expression cassette or recombinant bacterium for inhibiting plant viruses from infecting tobacco contains the nucleic acid sequence of the NbSRC2 gene, and the nucleic acid sequence of the NbSRC2 gene is shown in SEQ ID NO:1.

[0016] Advantages of the technical solution of the present invention

[0017] The present invention provides a tobacco antiviral protein gene NbSRC2, which is amplified from the model plant Nicotiana benthamiana. Laser confocal observation shows that the protein is mainly located in the cytoplasm, and expression pattern analysis shows that the protein gene is induced to express by virus infection and reaches the peak expression at 3dpi, with the highest expression in the root tissue of the plant. By silencing NbSRC2 through VIGS technology, it is jointly explained from three aspects of the difference in viral coat protein CP mRNA, coat protein CP and virus infection symptoms that silencing NbSRC2 upregulates the sensitivity of Nicotiana benthamiana to TMV infection and promotes virus infection. At the same time, the constructed overexpression vector 35S::NbSRC2::GFP was used to infiltrate Nicotiana benthamiana, which also proved that overexpression of NbSRC2 inhibited TMV infection. This shows that NbSRC2, as a new potential disease-resistant gene, can resist virus infection. The protein can inhibit the infection of plant viruses and is a potential antiviral gene on tobacco. NbSRC2 can be used as a potential antiviral protein, further enriching the scope of antiviral genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Relative expression of NbSRC2 mRNA in roots, stems, leaves and flowers of Nicotiana benthamiana (data were analyzed by Duncan multi-group sample difference analysis in SPSS ANOVA program, and different letters indicate significant differences in the values ​​of the four treatments (P<0.05));

[0019] Figure 2 The expression trend of TMV CP mRNA at 1, 2, 3, and 4 days after TMV inoculation (the data were analyzed by Duncan multi-group sample difference analysis in SPSS ANOVA program, and different letters indicate that the values ​​of the four treatments are significantly different (P<0.05));

[0020] Figure 3 The expression trend of NbSRC2 mRNA at 1, 2, 3, and 4 days after TMV inoculation (the data were analyzed by Duncan multi-group sample difference analysis in SPSS ANOVA program, and different letters indicate that the values ​​of the four treatments are significantly different (P<0.05));

[0021] Figure 4 Subcellular localization of NbSRC2;

[0022] Figure 5 The expression of TMV CP in different treatment groups at the RNA level after silencing NbSRC2 was different (the data were analyzed by independent sample t test, * indicates that the values ​​of the two treatments were significantly different (P<0.05));

[0023] Figure 6After silencing NbSRC2, 3 days after TMV infection, TMV CP protein levels were different among different treatment groups;

[0024] Figure 7 Silencing NbSRC2, 3 days after TMV infection, the symptoms of N. benthamiana in different treatment groups were different;

[0025] Figure 8 The expression of TMV CP in different treatment groups at the RNA level after overexpression of NbSRC2 was different (the data were analyzed by independent sample t test, * indicates that the values ​​of the two treatments were significantly different (P<0.05));

[0026] Fig. 9 Overexpression of NbSRC2, 3 days after TMV infection, differences in TMV CP protein levels between different treatment groups;

[0027] Fig.10 Overexpression of NbSRC2, 3 days after TMV infection, the symptoms of Nicotiana benthamiana in different treatment groups were different. DETAILED DESCRIPTION

[0028] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below in conjunction with specific examples and with reference to data. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0029] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The experimental materials, reagents, drugs, etc. used in the following examples, unless otherwise specified, can all be purchased through general channels.

[0030] In the following embodiments,

[0031] Wild Nicotiana benthamiana seeds were obtained from the Key Laboratory of Tobacco Pest and Disease Monitoring, Prevention and Integrated Management, Tobacco Research Institute, Chinese Academy of Agricultural Sciences.

[0032] TRV2 vector was kindly provided by Professor Liu Yule from Tsinghua University;

[0033] LBA4404 Agrobacterium was purchased from Beijing Bomed Gene Technology Co., Ltd.;

[0034] The vector pFu28-GFP was purchased from Wuhan Miaoling Biotechnology Co., Ltd.

[0035] Example 1

[0036] Cloning of NbSRC2 Gene from Nicotiana tabacum

[0037] Total RNA was extracted from Nicotiana benthamiana using TRIzol reagent, and the first-strand cDNA was synthesized using total RNA and reverse transcriptase. The synthesized cDNA was used as a template and PCR amplification was performed using specific primers of the NbSRC2 gene; the obtained PCR product was subjected to 1% agarose gel electrophoresis, and the gel was recovered and Sanger sequencing was performed. The obtained sequence is shown in SEQ ID NO: 1, and the amino acid sequence encoded by it is shown in SEQ ID NO: 2.

[0038] SEQ ID NO:1(5'→3')

[0039] ATGGCAACTGGTTCTTCCTCTCGCCCTCCATCCAAACCCTACGATCTTGAAATCACCGTCGTCTCCGCCAAGCACCTCAAAAATGTCAATTGGCATCACGGCGATCTCAAGCCCTATGTCATCTTTTGGGTCGACCCGGACAACAGGCGCGCCACTCAAGCTGATAATTCCAGCAATATCCGACCCGTTTGGAACGAGCGGTTCGTCCTCCATCTTCCCCAATCTCCTCACGATACTGTCCTCACTCTCGAGGTATTCCATTCCAAACCCTCAGAAACTTCCAAACCTTTAGTCGGTACACTTAGGATTCCGTTGAAGGAACTTGTTAACTTGGATGATCCAAACAAAATCAGAACTTTTGAGCTCCGACGACCCTCGGGCCGTCCTCATGGTAAGATCCGACTGAAGCTCGCTATTCTTGAATCTCCGAACCCCCAGGATTACCAAATACCCCCTCCTTCTAGCTATTATTACTCTACTGCCCCTCTGCCGCCTACCTATAGATCATATTCTCCTTCTCCGTACTCCTCATACCCTCCACCTCCTCCGCTGCCTGTAGTGGCAGCGCCGCCACCTACTTCTCCATCTTCACCGCCTCCTCCGGCACATTCGTATCCTTACGGCGGCTACTCTGATCTGTACGCCAGTTACTACCCCGGGTACTACTCTCAGCCACCTCCTCCTCCTCGACCATTCGTTGAGCGGCAATCTAGCTATGGTGGGGGGCTGGGGTCGAGACCGAGTGCCCCTGCTGATTATCCTCCTAATTATGATCAGAAGCGTAGTGGAAAGATGGGAATGGGCACCGGATTGGCAGTTGGAACAGTGGCTGGCGTGCTCGGAGGATTGGCACTGGGGGAAGGGTTGAAGTACGAGGAAGAGAAGATTGCTGGGAGAGTTGAAGACAACATAGCCTCCAGGGATGATTACAGCAATTACAGTGTTGAATATTGA

[0040] SEQ ID NO:2

[0041] MATGSSRPPSKPYDLEITVVSAKHLKNVNWHHGDLKPYVIFWVDPDNRRATQADNSSNIRPVWNERFVLHLPQSPHDTVLTLEVFHSKPSETSKPLVGTLRIPLKELVNLDDPNKIRTFELRRPSGRPHGKIRLKLAILESPNPQDYQIPPPSSYYY STAPLPPTYRSYSPSPYSSYPPPPPLPVVAAPPPTSPSSPPPPAHSYPYGGYSDLYASYYPGYYSQPPPPPRPFVERQSSYGGGLGSRPSAPADYPPNYDQKRSGKMGMGTGLAVGTVAGVLGGLALGEGLKYEEEKIAGRVEDNIASRDDYSNYSVEY

[0042] The specific primer sequences for amplifying the NbSRC2 gene are as follows:

[0043] F: 5'-ATGGCAACTGGTTCTTCCTC-3' (SEQ ID NO: 3);

[0044] R: 5'-ATATTCAACACTGTAATTGCTGT-3' (SEQ ID NO: 4).

[0045] Example 2

[0046] Expression pattern and subcellular localization of tobacco NbSRC2 gene

[0047] (1) Expression pattern

[0048] Total RNA of tobacco was extracted, and cDNA samples were synthesized according to the procedure of the reverse transcription kit (Vazyme, Nanjing, China). The relative expression level of mRNA of the target gene was quantitatively detected according to the instructions of the real-time fluorescence quantitative PCR kit (Vazyme, Nanjing, China). In order to detect the relative expression of NbSRC2 in different tissues of tobacco, samples were taken from the roots, stems, leaves and flowers of tobacco. In order to monitor the expression trend of NbSRC2 under the stress of virus infection, Nicotiana benthamiana was inoculated with TMV virus juice, and samples were taken to detect the relative expression levels of TMV CP and NbSRC2 mRNA 1, 2, 3, and 4 days after inoculation. Primer sets TMV-F / TMV-R, SRC2-F / SRC2-R and β-ActinQF / β-ActinQR were used to amplify TMV CP, NbSRC2 and β-actin, respectively. 2 -ΔΔCtThe relative expression levels of these genes were calculated by the PCR method. All experiments were repeated three times with three biological replicates.

[0049] qRT-PCR analysis results showed that NbSRC2 expression was detected in various tissues of wild-type Nicotiana benthamiana, and the transcript level of NbSRC2 in root tissue was higher than that in other tissues tested ( Figure 1 ); TMV CP mRNA expression was detected at 2 dpi after TMV inoculation, and showed an increasing trend with the inoculation time ( Figure 2 ); TMV infection induced the expression of NbSRC2 at 1 dpi and reached the maximum at 3 dpi ( Figure 3 ).

[0050] (2) Subcellular localization

[0051] To construct the p35S::SRC2::GFP expression vector, the NbSRC2 CDS sequence was first cloned into the intermediate vector pFu28-GFP by homologous recombination and then cloned into pEarleygate by LR reaction. The constructed p35S::SRC2::GFP expression plasmid was transformed into Agrobacterium tumefaciens LBA4404. The transformed Agrobacterium tumefaciens was cultured in a buffer containing 10 mmol / L MES, 200 μmol / L AS and 10 mmol / L MgCl2 until the OD 600 The p35S::SRC2::GFP Agrobacterium was used to infiltrate the back of Nicotiana benthamiana leaves using a needleless syringe. After 48 hours, the subcellular localization of the target protein NbSRC2 was observed under laser confocal microscopy. The results showed that the protein was mainly localized in the cytoplasm ( Figure 4 ).

[0052] Example 3

[0053] Effects of silencing NbSRC2 on plant virus infection of Nicotiana benthamiana

[0054] (1) Construction of NbSRC2 silencing vector:

[0055] The NbSRC2 gene fragment was used as a template and the following primer pair was used for amplification to obtain a 241 bp gene fragment; the TRV2 vector was double-digested with (EcoRI) / (KpnI) and then ligated with the above PCR amplification product to obtain the silencing vector pSRC2::TRV2, which was then transformed into LBA4404 Agrobacterium.

[0056] F: 5'-TAAGGTTACCGAATTCTGCCGCCTACCTATAGATCATATTC-3' (SEQ ID NO: 5);

[0057] R: 5'-AGACGCGTGAGCTCGGTACCCGTAAGGATATGAATGTGCCGGA-3' (SEQ ID NO: 6);

[0058] (2) The LBA4404 Agrobacterium containing the silencing vector pSRC2::TRV2 was used to infiltrate Nicotiana benthamiana.

[0059] (3) Infect the Nicotiana benthamiana treated in step (2) with TMV as the treatment group TRV::SRC2; and infect the Nicotiana benthamiana not treated in step (2) with TMV as the control group TRV::00; and test the virus infection for at least two weeks. The results showed that 1-4 days after TMV infection, the viral CP expression level of the treatment group TRV::SRC2 was significantly higher than that of the control group, which was 3.4 times and 5.1 times that of the control group at 2 and 3 dpi, respectively ( Figure 5 At 3 dpi, protein immunoblotting detected that the viral CP protein level in the treatment group TRV::SRC2 was higher than that in the control group TRV::00 ( Figure 6 At 3 dpi, the treated group showed obvious symptoms of virus infection (tops wilted and fell, Figure 7 ). Taken together, these results indicate that silencing NbSRC2 renders N. benthamiana highly susceptible to plant virus infection.

[0060] Example 4

[0061] Effects of overexpression of NbSRC2 on plant virus infection of Nicotiana benthamiana

[0062] 1. Construction of overexpression vector p35S::SRC2::GFP

[0063] Using the NbSRC2 gene fragment as a template, PCR amplification was performed using the following primer pairs to obtain PCR products;

[0064] F: 5'-CTTTAGATCTTCTAGAATGGCAACTGGTTCTTCCTC-3' (SEQ ID NO: 7);

[0065] R: 5'-CCATGAGCTCGAATTCATATTCAACACTGTAATTGCTGT-3' (SEQ ID NO: 8);

[0066] The vector pFu28-GFP was double-digested with XbaI / EcoRI and then ligated with the above PCR product to obtain the plant overexpression vector p35S::SRC2::GFP.

[0067] 2. The constructed p35S::SRC2::GFP expression vector plasmid was used to transform into Agrobacterium tumefaciens LBA4404. The transformed Agrobacterium tumefaciens was cultured in a buffer containing 10 mmol / L MES, 200 μmol / L AS and 10 mmol / L MgCl2 until the OD 600 Reaching 0.8, instantly soaking the tobacco leaves.

[0068] 3. Infect Nicotiana benthamiana treated in step (2) with TMV as the treatment group 35S::SRC2 / TMV; and infect Nicotiana benthamiana not treated in step (2) with TMV as the control group 35S::00 / TMV; detect the difference in virus expression between the treatment and control groups from RNA, protein and virus infection symptoms. Samples were taken at 1, 2, and 3 days after inoculation, and the expression of TMV CP at the RNA level was detected by fluorescence quantitative PCR. The results are as follows Figure 8 The expression of the virus in the treatments shown was significantly lower than that in the control. The samples taken on the 3rd day were subjected to a protein immunoblotting test, and the results were consistent with those at the gene level ( Fig. 9 At 3 dpi, the control group 35S::00 / TMV inoculated with TMV showed obvious symptoms of virus infection ( Fig.10 ). In summary, overexpression of NbSRC2 inhibits the infection of plant viruses.

[0069] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A tobacco antiviral protein gene NbSRC2, characterized in that: The amino acid sequence encoded by the gene is shown in SEQ ID NO:

2.

2. The tobacco antiviral protein gene NbSRC2 according to claim 1, characterized in that: The nucleic acid sequence of the gene is shown in SEQ ID NO:

1.

3. Use of the tobacco antiviral protein gene NbSRC2 according to claim 1 or 2 in inhibiting plant viruses from infecting tobacco.

4. The use according to claim 3, characterized in that: The plant virus is at least one of tobacco common mosaic virus, tobacco cucumber mosaic virus and potato virus Y.

5. A method for inhibiting plant viruses from infecting tobacco, characterized in that: The gene sequence of NbSRC2 is constructed into an expression vector, and tobacco is transformed to make it overexpressed in tobacco, thereby inhibiting plant viruses from infecting tobacco; the nucleic acid sequence of the NbSRC2 gene is shown in SEQ ID NO:

1.

6. The method for inhibiting plant viruses from infecting tobacco according to claim 5, characterized in that: The expression vector is any one of a prokaryotic expression vector, a eukaryotic expression vector and a viral expression vector.

7. The method for inhibiting plant viruses from infecting tobacco according to claim 5, characterized in that: The plant virus is at least one of tobacco common mosaic virus, tobacco cucumber mosaic virus and potato virus Y.

8. The method for inhibiting plant viruses from infecting tobacco according to any one of claims 5 to 7, characterized in that: The plant virus is tobacco common mosaic virus.

9. A recombinant vector, expression cassette or recombinant bacterium for inhibiting plant viruses from infecting tobacco, characterized in that: Contains the nucleic acid sequence of NbSRC2 gene, and the nucleic acid sequence of NbSRC2 gene is shown in SEQ ID NO:1.

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