Application of GmC2H2 protein or its encoding gene in plant antiviral

Abstract

The present invention discloses the application of the GmC2H2 protein or its coding gene in plant antiviral. By means of Agrobacterium-mediated transformation, the present invention transfers the GmC2H2 gene into the target plant, and obtains a method for obtaining a plant overexpressing the soybean GmC2H2 gene. The obtained soybean GmC2H2 overexpressing plant can significantly reduce the infection of soybean mosaic virus. The stable overexpression of the GmC2H2 gene in soybean and the transient overexpression of the GmC2H2 gene in tobacco can endow plants with resistance.

Description

Technical Field

[0001] The present invention belongs to the application field of agricultural science and technology, and particularly relates to the application of GmC2H2 protein or its encoding gene in plant antiviral. Background Art

[0002] Soybean mosaic virus (SMV) is one of the most common viral diseases in the world's soybean production areas. There are many factors contributing to the prevalence of soybean mosaic virus disease, mainly including seed-borne virus and aphid transmission in the field. Susceptible soybean germplasms are vulnerable to soybean mosaic virus, resulting in reduced yield and deteriorated quality. Diseased seedlings grown from virus-infected seeds are the primary infection sources for the prevalence of soybean mosaic virus. The number of virus-transmitting insects is the dominant factor affecting the field environment. In particular, aphids that transmit viruses in a non-persistent manner are likely to cause the spread of SMV, leading to large-scale prevalence of the disease in the field.

[0003] In addition to directly causing severe soybean yield reduction and germplasm decline, the infection of SMV also greatly reduces the immunity of soybeans to other pathogens. Therefore, SMV has become the main factor restricting soybean production. Currently, there is no safe and effective chemical agent to control soybean mosaic virus disease. Exploring disease-resistant genes and cultivating disease-resistant varieties are the most economical and effective methods for controlling soybean mosaic virus disease.

[0004] Zinc finger proteins (ZFPs) are a class of nucleic acid-binding proteins that play important roles in eukaryotic cells most widely. Zinc finger proteins can be divided into 9 subfamilies according to their conserved Cys and His motifs, covering C2H2, C3H, C3HC4, C2HC5, C4HC3, CCCH, C4, C6, and C8 subfamilies. C2H2-type zinc finger proteins are generally present in plants, and there are 321 of them in soybeans. Currently known plant C2H2-type zinc finger proteins mainly participate in various stages of plant growth and development. They are involved in the regulation of gene expression under environmental stress. For example, the NSG1 gene is involved in regulating the development of spikelets in rice (Zhuang et.al, 2020), the ZFP245 gene is involved in regulating the response to adversity stress (Huang et al., 2005), the ONAC022 gene is involved in regulating the response to drought and salt stress in rice (Hong et al., 2016), and the GmbZIP1 gene has been found to be involved in regulating tolerance under drought stress (Gao et al., 2011). There is currently no research report on the antiviral activity of plant C2H2 proteins. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide the application of GmC2H2 protein or the GmC2H2 gene encoding the protein in plant antiviral.

[0006] Another technical problem to be solved by the present invention is to provide the application of an overexpression vector containing the disease-resistant gene GmC2H2 or a strain containing the overexpression vector in plant antiviral.

[0007] Another technical problem to be solved by the present invention is to provide a method for obtaining antiviral plants.

[0008] The last technical problem to be solved by the present invention is to provide a method for identifying whether a plant obtained by the described method has antiviral ability.

[0009] Technical solution: To solve the above technical problems, the present invention provides the application of GmC2H2 protein or the GmC2H2 gene encoding the protein in plant antiviral. The amino acid sequence of the GmC2H2 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the GmC2H2 gene is shown in SEQ ID NO.1.

[0010] Among them, the application includes enhancing the resistance of plants to viruses by overexpressing the expression level and / or activity of GmC2H2 protein.

[0011] Among them, the method of overexpressing the expression level and / or activity of GmC2H2 protein is achieved by overexpressing the GmC2H2 gene.

[0012] The present invention also includes the application of an overexpression vector containing the GmC2H2 gene or a strain containing the overexpression vector in plant antiviral. The nucleotide sequence of the GmC2H2 gene is shown in SEQ ID NO.1.

[0013] Among them, the plants include but are not limited to soybeans or tobacco.

[0014] The present invention also includes a method for obtaining antiviral plants. The method includes: upregulating the expression or activity of GmC2H2 protein, and the amino acid sequence of the GmC2H2 protein is shown in SEQ ID NO.2.

[0015] Among them, the method of upregulating the expression or activity of GmC2H2 protein is to overexpress the GmC2H2 gene in the plant genome, and the nucleotide sequence of the GmC2H2 gene is shown in SEQ ID NO.1.

[0016] Among them, the virus is one or two of soybean mosaic virus (SMV) or turnip mosaic virus belonging to the genus Potyvirus.

[0017] The present invention also includes a method for identifying whether a plant obtained by the described method has antiviral ability, by identifying whether the plant contains a C2H2 protein or a C2H2 gene.

[0018] Among them, the primer sequences for detecting the target of soybean mosaic virus of the genus Potyvirus are shown in SEQ ID NO.5 and SEQ ID NO.6, and the primer sequences for detecting the target of turnip mosaic virus are shown in SEQ ID NO.7 and SEQ ID NO.8. Among them, the plants include soybean or Nicotiana benthamiana.

[0019] Among them, the identification method is detected by RT-qPCR or Western blotting.

[0020] Beneficial effects: Compared with the prior art, the outstanding effect of the present invention is that the present invention first discovers that the stable overexpression of the GmC2H2 gene in soybean and the transient overexpression of the GmC2H2 gene in tobacco can endow plants with resistance.

[0021] The following further describes the application of the overexpressed plant C2H2 in regulating plant antiviral and the method for cultivating transgenic plants according to the present invention in conjunction with the accompanying drawings and specific implementation methods. Description of the Drawings

[0022] Figure 1 Transient overexpression of GmC2H2 confers resistance of Nicotiana benthamiana to soybean mosaic virus (SMV). After inoculating Nicotiana benthamiana with SMV-GFP and YFP empty vector, and SMV-GFP and GmC2H2 respectively for 60 hours, the SMV RNA level in the plants was detected by RT-qPCR.

[0023] Figure 2 Transient overexpression of GmC2H2 confers resistance of Nicotiana benthamiana to turnip mosaic virus (TuMV). After inoculating Nicotiana benthamiana with TuMV-GFP and YFP empty vector, and TuMV-GFP and GmC2H2 respectively for 36 hours and 48 hours, the TuMV RNA level in the plants was detected by RT-qPCR.

[0024] Figure 3 PCR detection of the regenerated plants of Williams 82 transformed with GmC2H2-pFGC5941. M: Marker; +: Positive control GmC2H2-pFGC5941 vector; -: Negative control; 1-8: Amplification results of positive transgenic plants.

[0025] Figure 4Overexpression of GmC2H2 confers resistance to soybean mosaic virus (SMV) in soybean. (A) Phenotypes of WT and GmC2H2 overexpressing plants at 10 days post inoculation (dpi) with SMV. WT plants showed mosaic symptoms, while GmC2H2 transgenic plants showed milder symptoms; (B) RT-qPCR analysis of SMV RNA levels in plants; (C) Western blotting analysis of SMV protein accumulation in the above plants; CBB represents Coomassie Brilliant Blue, α-CP is the coat protein, and RbcL is the internal reference gene. Detailed implementation mode

[0026] The following will describe the implementation scheme of the present invention in detail in combination with examples. The following examples are only used to illustrate the present invention. Those not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer can be obtained as conventional products through commercial purchase.

[0027] Example 1 Transient overexpression of GmC2H2 in Nicotiana benthamiana confers resistance to virus

[0028] Soybean Williams82 RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, the primer pair was: 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGACAGATCCTAAGTCCAAT-3’ (SEQ ID NO.3) and 5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCCTCCATCATGTTCAATTTCAG-3’ (SEQ ID NO.4). The resulting PCR product was GmC2H2. The PCR reaction system was 25 μL, including 12.5 μL of PCR Mix, 10 ng of cDNA template, 1 μL of each primer, and water was added to make up 25 μL. The reaction conditions were: pre-denaturation at 94 °C for 3 min; 94 °C for 30 s, 57 °C for 30 s, 72 °C for 1 min, for 30 cycles; finally, extension at 72 °C for 7 min. Using the Gateway vector construction method (Invitrogen), GmC2H2 was ligated to the vector pEarleyGate104 (purchased from TAIR) with a YFP fluorescent tag to obtain the GmC2H2-YFP recombinant plasmid. 1 μg of the above GmC2H2-YFP recombinant plasmid was mixed with 100 μl of Agrobacterium competent cells (GV3101 strain) and transferred into a clean electroporation cup. Electroporation was carried out at 2500 V. Immediately, pre-cooled LB liquid medium was added. After recovery at 28 °C for 2 h, it was spread on the resistant medium and grown for 48 h to screen for positive Agrobacterium.

[0029] To confirm the role of the GmC2H2 gene in virus infection, GmC2H2-YFP and SMV-GFP (Yin J, Liu H, Xiang W, Jin T, Guo D, Wang L, Zhi H. Discovery of the Agrobacterium growth inhibition sequence in virus and its application to recombinant clone screening. AMB Express. 2019 Jul 24;9(1):116. doi: 10.1186 / s13568-019-0840-3. PMID: 31342207; PMCID: PMC6656845.) were transiently co-expressed in Nicotiana benthamiana leaves. The specific method was to culture Agrobacterium containing GmC2H2-YFP, SMV-GFP, and the pEarleyGate104 empty vector overnight at 28 °C and 200 rpm respectively. Adjust the OD600 of each Agrobacterium to 1.2, and then mix the Agrobacterium of SMV-GFP and the pEarleyGate104 empty vector (control group), and SMV-GFP and GmC2H2-YFP at a ratio of 1:1 respectively, and inject them into the leaves of 3- to 4-week-old Nicotiana benthamiana. After 60 hours of inoculation, take the inoculated leaves to extract RNA, and detect the viral RNA in the inoculated leaves by RT-qPCR. The detection primer pairs were: 5’-GAACACAGTTCGAAGCGTGG-3’ (SEQ ID NO.5) and 5’-ACGCCATTTGCATCTGGAGA-3’ (SEQ ID NO.6). The results showed that the accumulation of viral RNA in the samples co-expressing GmC2H2-YFP was extremely significantly lower than that in the control group ( Figure 1 ), indicating that overexpression of GmC2H2 can make the plants resistant to viruses. Using the same method, GmC2H2 and TuMV-GFP were transiently co-expressed in Nicotiana benthamiana. After 36 hours and 48 hours of inoculation, take the inoculated leaves to extract RNA, and detect the viral RNA in the inoculated leaves by RT-qPCR. The detection primer pairs were: 5’-GGCACTCAAGAAAGGCAAGG-3’ (SEQ ID NO.7) and 5’-CTCCGTCAGTTCGTAATCAGC-3’ (SEQ ID NO.8). The results showed that the accumulation of viral RNA in the samples overexpressing GmC2H2-YFP was extremely significantly lower than that in the control group ( Figure 2 ).

[0030] The above results indicate that transient overexpression of GmC2H2 in tobacco confers resistance to different viruses (SMV and TuMV) of the Potyvirus genus.

[0031] Example 2: Overexpression of GmC2H2 in soybean confers virus resistance to plants

[0032] Soybean Williams82 RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, the GmC2H2 gene fragment was amplified by PCR with primers. Restriction enzyme cleavage sites of XhoI and SmaI and homologous arms were added to the forward and reverse primers respectively. The primer pair was: 5’-TTTGGAGAGGACACGCTCGAG ATGACAGATCCTAAGTCCAATGTAT-3'(SEQ ID NO.9) and 5’-GTATGGGTACATGGGCGCCG TCCTCCATCATGTTCAATTTCAG-3'(SEQ ID NO.10). The amplified fragment was ligated into the pFGC5941 vector (purchased from TAIR) digested with XhoI and SmaI (37 °C, 2 h), and the overexpression vector of pFGC5941-GmC2H2 could be obtained. 10 μl of the above ligation product was added to 50 μl of Escherichia coli competent cells (DH5α strain) and mixed evenly. It was incubated on ice for 30 min, in a 42 °C water bath for 60 s, and on ice for 2 min successively. Immediately, non-resistant LB liquid medium was added, and after recovery at 37 °C for 1 h, it was spread on the resistant medium and cultured overnight. Samples identified as positive by colony PCR were selected to extract plasmids and then sent for sequencing. 1 μl of the overexpression vector plasmid of pFGC5941-GmC2H2 was added to 50 μl of Agrobacterium competent cells (GV3101 strain) and mixed evenly. It was incubated on ice, in liquid nitrogen, in a 37 °C water bath, and on ice for 5 min successively. Immediately, non-resistant LB liquid medium was added, and after recovery at 28 °C for 2 h, it was spread on the resistant medium and grown for 48 h, and positive Agrobacterium was screened and obtained.

[0033] The above positive single colony was transferred into 5 ml of liquid resistant medium (LB medium resistant to rifampicin Rif and kanamycin Kan), and cultured at 28 °C and 100 rpm for 24 h. Then, these 5 ml of bacterial strains were transferred to 100 ml of liquid resistant medium and cultured at 28 °C and 100 rpm for 12 h. The OD600 of Agrobacterium was adjusted to reach 0.6 - 1.0. The bacterial liquid was centrifuged at 3600 revolutions per

[0034] Centrifuge for 10 min at 25°C, pour off the supernatant, suspend the bacterial pellet in liquid resistant medium, adjust the Agrobacterium OD600 to 0.8, inoculate soybean Williams82, obtain the infected soybean cotyledon nodes, differentiate and culture to obtain clustered buds, screen and culture to obtain resistant buds, then obtain seedlings through rooting culture, harden off the seedlings and transplant them for continuous culture to obtain T0 generation regenerated soybean plants. Take some leaf samples, extract the DNA of the regenerated soybean plants by CTAB method, use this as a template, and amplify a 1029 bp fragment using primer pairs SEQ ID NO.9 and SEQ ID NO.10, and sequence and compare to detect whether the regenerated plants are positive. After sequencing, 8 positive plants were obtained ( Figure 3 ).

[0035] Friction-inoculate SMV on wild-type soybean (Williams 82) and GmC2H2 transgenic soybean respectively. After 10 days, typical mosaic symptoms appeared in wild-type plants, but the symptoms of transgenic soybean were milder ( Figure 4 A). RT-qPCR (primer pairs SEQ ID NO.5 and SEQ ID NO.6) and western blotting results showed that both viral RNA and coat protein α-CP in GmC2H2 overexpressing soybeans were significantly lower than those in wild-type soybeans ( Figure 4 B and Figure 4 C), indicating that GmC2H2 overexpression confers resistance to soybeans.

[0036] Collectively, the above results indicate that overexpression of C2H2 confers resistance to viruses in plants (tobacco and soybean).

Claims

1. GmC2H2 protein or a protein encoding the same GmC2H2 The application of the gene in plant antiviral, the amino acid sequence of the GmC2H2 protein is shown in SEQ ID NO. 2, the GmC2H2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, the plant is soybean or tobacco, and the virus is one or both of soybean mosaic virus (SMV) or turnip mosaic virus of the genus Potyvirus.

2. The use according to claim 1, characterized in that: The application includes enhancing the resistance of plants to viruses by increasing the expression level and / or activity of the GmC2H2 protein.

3. The use according to claim 2, characterized in that: The method for increasing the expression level and / or activity of the GmC2H2 protein is by overexpressing GmC2H2 Genes to achieve.

4. Contains GmC2H2 Application of a gene overexpression vector or a strain containing the overexpression vector in plant anti-virus, the GmC2H2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1, the plant is soybean or tobacco, and the virus is one or both of soybean mosaic virus (SMV) or turnip mosaic virus (turnipmosaic virus) of the genus Potyvirus.

5. A method for obtaining virus-resistant plants, characterized in that: The method comprises: up-regulating the expression or activity of GmC2H2 protein, the amino acid sequence of the GmC2H2 protein is shown in SEQ ID NO. 2, the plant is soybean or tobacco, and the virus is one or both of soybean mosaic virus (SMV) or turnip mosaic virus of the genus Potyvirus.

6. The method according to claim 5, characterized in that The method for upregulating the expression or activity of the GmC2H2 protein is to overexpress in the genome of the plant GmC2H2 Gene, GmC2H2 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

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