Use of gmhb6 protein or its encoding gene in plant virus resistance

Abstract

The application discloses application of a soybean GmHB6 protein or an HB6 gene coding the protein in plant antiviral activity. The application introduces the GmHB6 gene into a target plant by a method of agrobacterium transformation, and obtains a soybean GmHB6 gene overexpression plant. The soybean GmHB6 overexpression plant obtained by the method of the application can significantly reduce infection of a soybean mosaic virus.

Description

Technical Field

[0001] This invention belongs to the field of agricultural science and technology, specifically relating to the application of GmHB6 protein or its encoding gene in plant antiviral activity. Background Technology

[0002] Soybean mosaic virus (SMV) is a global disease affecting soybean-producing regions worldwide. The first report in my country dates back to 1899 in the Northeast soybean-producing region, followed by reports from soybean-producing areas across the country, including Shandong, Jiangsu, and Hubei. Generally, soybean mosaic virus causes yield losses of 15-35%, but this varies depending on factors such as the planting season, variety resistance, SMV infection stage, and virus strain. In some severely affected years, yield losses can exceed 70%, even leading to total crop failure (Ren et al., 1997).

[0003] Homeodomain-leucine zipper (HDZip) proteins are plant-specific transcription factors containing a typical homeodomain-leucine zipper (HD-Zip) structure. These proteins are present in various plant tissues and organs, possessing multiple functions and serving as important regulators of growth, development, and abiotic stress responses in higher plants. Increasing evidence suggests that HD-Zip I family proteins are involved in plant responses to abiotic stress (Gong et al., 2019), and their stress-induced expression, such as OsMYB3R-2 (Dai et al., 2007), OsSDIR1 (Gao et al., 2011), ABF2, and SlHZ24 (Hu et al., 2016), enhances plant adaptability to abiotic stress. Some involve changes in plant morphology, including leaf development and root hair formation (Aoyama et al. 1995; Di Cristina 1996; Ohgishi et al. 2001). Overexpression of the Arabidopsis ATHB13 gene can lead to developmental abnormalities such as smaller leaves and less distinct connection between petioles and leaves (Hanson et al., 2001).

[0004] There are currently no research reports on the antiviral properties of plant HB6 protein, and there is an urgent need to explore genes containing leucine zipper structures for antiviral applications. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an application of the soybean leucine zipper GmHB6 gene in regulating plant antiviral activity.

[0006] The technical problem to be solved by the present invention is to provide a method for obtaining antiviral plants.

[0007] The technical problem to be solved by the present invention is to provide a method for determining whether a plant obtained by the method has antiviral capabilities.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides the application of GmHB6 protein or GmHB6 gene encoding said protein in plant antiviral activity. The amino acid sequence of said GmHB6 protein is shown in SEQ ID NO.2, and the nucleotide sequence of said GmHB6 gene is shown in SEQ ID NO.1.

[0009] The present invention also includes a method for obtaining antiviral plants, the method comprising: upregulating the expression or activity of GmHB6 protein, wherein the amino acid sequence of GmHB6 protein is shown in SEQ ID NO.2.

[0010] The method for upregulating the expression or activity of GmHB6 protein is to overexpress the GmHB6 gene in the plant genome, and the nucleotide sequence of the GmHB6 gene is shown in SEQ ID NO.1.

[0011] The virus in question is either soybean mosaic virus (SMV) or turnip mosaic virus, belonging to the genus Potato virus Y.

[0012] The plants mentioned include, but are not limited to, soybeans or Nicotiana benthamiana.

[0013] The present invention also includes a method for identifying whether the plant obtained by the method has antiviral ability, by identifying whether the plant contains HB6 protein or HB6 gene.

[0014] The virus in question is either soybean mosaic virus or turnip mosaic virus, belonging to the genus PotatoY.

[0015] The primer sequences for detecting the target of soybean mosaic virus (Potatovirus Y) 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.

[0016] The identification method involves detection via RT-qPCR or Western blotting.

[0017] Beneficial Effects: Compared with existing technologies, the outstanding effect of this invention is that it is the first discovery that soybean GmHB62 is a disease resistance gene. By transforming the HB6 gene into target plants using Agrobacterium, the resulting soybean plants with upregulated HB6 expression possess resistance to SMV, effectively controlling the damage caused by this virus. The stable overexpression of the GmHB6 gene in soybean and the transient overexpression of the GmHB6 gene in tobacco obtained by this invention can induce plant resistance. Attached Figure Description

[0018] Figure 1 This is a PCR electrophoresis image showing the process of constructing the GmHB6-pBA002 vector. M: Marker; 1: HB6 amplification results in the GmHB6-pBA002 vector.

[0019] Figure 2 Transient overexpression of GmHB6 induced resistance to soybean mosaic virus (SMV) in Nicotiana benthamiana. After inoculating Nicotiana benthamiana with SMV-GFP and empty GFP vectors, and SMV-GFP and GmHB6 vectors, respectively, for 60 hours, the SMV RNA levels in the plants were detected by RT-qPCR.

[0020] Figure 3 Transient overexpression of GmHB6 induced resistance to turnip mosaic virus (TuMV) in Nicotiana benthamiana. TuMV-GFP and empty GFP vectors, and TuMV-GFP and GmHB6 were inoculated into Nicotiana benthamiana for 48 hours and 72 hours, respectively, and the TuMV RNA levels in the plants were detected by RT-qPCR.

[0021] Figure 4 Overexpression of GmHB6 induced resistance to soybean mosaic virus (SMV) in soybeans. (A) Phenotypes of WT and GmHB6-overexpressing plants 10 days after SMV inoculation. WT plants also showed mosaic symptoms, while GmHB6 transgenic plants showed milder symptoms; (B) RT-qPCR detection of SMV RNA levels in plants; (C) Western blotting detection of SMV protein accumulation in the above plants. Detailed Implementation

[0022] The embodiments of the present invention will be described in detail below with reference to the examples. The following examples are only for illustrating the present invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0023] Example 1: Transient overexpression of GmHB6 in Nicotiana benthamiana induces virus resistance in plants.

[0024] RNA was extracted from Williams82 soybeans and reverse transcribed to obtain cDNA. Using the cDNA as a template, Xbal and SmaI restriction enzyme sites and homologous arms were added to the 5' and 3' ends of GmHB6 (the amino acid sequence of the GmHB6 protein is shown in SEQ ID NO.2, and the nucleotide sequence of the GmHB6 gene is shown in SEQ ID NO.1), respectively. The primer pairs used were: 5'-ACGATAGCCGGATCATCTAGAATGAAGAGACTTGGCAGTTCG-3' (SEQ ID NO.3) and 5'-ATCGAATTCCTGCAGCCCGGGACTCCATTCCTCTGAACAGTACC-3' (SEQ ID NO.4). The PCR reaction volume was 25 μL, including 12.5 μL of PCR Mix, 10 ng of cDNA template, 1 μL of each primer, and water to make up to 25 μL. The reaction conditions were: 94℃ pre-denaturation for 3 min; 94℃ for 30 s, 57℃ for 30 s, 72℃ for 1 min, 30 cycles; and a final extension at 72℃ for 7 min. The resulting PCR product was the GmHB6 gene fragment. Figure 1 Using a homologous recombination vector construction method, the GmHB6 gene fragment was ligated into the vector pGD-C-EGFP with a GFP fluorescent tag (this vector was provided by the authors of the article, Zhang Y, Chai M, Cheng X, Xu K. Transiently Induce RNA Silencing in Plants Using a Tobacco Necrosis Virus A(TNV-A)-Based dsRNA Production System. Methods Mol Biol. 2024; 2771:83-89. doi:10.1007 / 978-1-0716-3702-9_12.PMID:38285394.), to obtain the GmHB6-GFP recombinant plasmid. Mix 1 μg of the above GmHB6-GFP recombinant plasmid with 50 μL of Agrobacterium competent cells (GV3101 strain), and incubate in ice bath, liquid nitrogen, 37℃ water bath, and ice bath for 5 min in sequence. Immediately add antibiotic-free LB liquid medium, recover at 28℃ for 2 hours, and then spread onto antibiotic medium for 48 hours of growth. Positive Agrobacterium strains are obtained by screening.

[0025] To confirm the role of HB6 in viral infection, transiently expressed GmHB6-GFP and SMV-GFP (the viral vector was provided by the authors of the article, 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 co-infected in tobacco leaves. The specific method involved culturing Agrobacterium containing GmHB6-GFP, SMV-GFP, and empty GFP vectors overnight at 28℃ and 200rpm, respectively, adjusting the OD600 of each Agrobacterium to 1.2. Then, Agrobacterium containing SMV-GFP and the empty pGD-C-EGFP vector (as the control group) and SMV-GFP and GmHB6-GFP (as the experimental group) were mixed 1:1 and injected into leaves of 3- to 4-week-old *Tobacco Bengal* plants. Sixty hours after inoculation, RNA was extracted from the inoculated leaves, and RT-qPCR was used to detect viral RNA in the inoculated leaves. 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 viral RNA accumulation in the samples co-expressing GmHB6-GFP was significantly lower than that in the control group. Figure 2This indicates that overexpression of GmHB6 can enhance plant resistance to the virus. Using the same method, GmHB6 and TuMV-GFP (the viral vector is derived from the article: Xiaoyan Cui, Hoda Yaghmaiean, Guanwei Wu, Xiaoyun Wu, Xin Chen, Greg Thornc and Aiming Wang. 2017. The C-terminal region of the Turnip mosaic virus P3 protein is essential for viral infection via targeting P3 to the viral replication complex. Virology. 510:147-155.) were transiently expressed in *Nicotiana benthamiana* for co-infection. RNA was extracted from inoculated leaves at 48 and 72 hours after inoculation, and viral RNA was detected by RT-qPCR using the primer pairs: 5'-GGCACTCAAGAAAGGCAAGG-3' (SEQ ID NO.7) and 5'-CTCCGTCAGTTCGTAATCAGC-3' (SEQ ID NO.8). The results showed that the viral RNA accumulation in the sample overexpressing GmHB6-GFP was significantly lower than that in the control group. Figure 3 ).

[0026] The results above indicate that transient overexpression of GmHB6 in tobacco induces resistance to different viruses (SMV and TuMV) of the Potyvirus genus.

[0027] Example 2: Overexpression of GmHB6 in soybeans induces virus resistance in plants.

[0028] Soybean Williams82 RNA was extracted and reverse transcribed to obtain cDNA. Using the cDNA as a template, the GmHB6 gene fragment was amplified by PCR using primer pairs. XhoI restriction enzyme sites and homologous arms were added to the forward and reverse primers, respectively.

[0029] The primer pairs are: 5'-ACGGGGGACTCTAGAGGATCTCGAGATGAAGAGACTTGGCAGTTCG-3' (SEQ ID NO. 9) and 5'-GGCCTGGGCCCGGCGCGCCTCGAGTTAACTCCATTCCTCTGAACAGT-3' (SEQ ID NO. 10). The amplified fragment is ligated into the PBA002 vector digested with XhoI (37℃, 2h) (this vector was provided by Nanjing Agricultural University, see Chinese patent application with publication number CN117604003A) to obtain the overexpression vector of pBA002-GmHB6. 10 μL of the ligation product was added to 50 μL of competent *E. coli* cells (DH5α strain) and mixed thoroughly. The mixture was then incubated on ice for 30 min, in a 42°C water bath for 60 s, and on ice for 2 min. Immediately afterward, antibiotic-free LB broth was added. After recovery at 37°C for 1 hour, the mixture was plated onto antibiotic-resistant medium and incubated overnight. Samples identified as positive by bacterial PCR were selected, and plasmids were extracted and sent for sequencing. 1 μL of the pBA002-GmHB6 overexpression vector plasmid was added to 50 μL of competent *Agrobacterium* cells (GV3101 strain) and mixed thoroughly. The mixture was then incubated on ice, in liquid nitrogen, in a 37°C water bath, and on ice for 5 min. Immediately afterward, antibiotic-free LB broth was added. After recovery at 28°C for 2 hours, the mixture was plated onto antibiotic-resistant medium and incubated for 48 hours. Positive *Agrobacterium* samples were obtained through screening.

[0030] The above-mentioned positive single colonies were transferred to 5 mL of liquid resistance medium and cultured at 28°C and 100 rpm for 24 h. This 5 mL of inoculum was then transferred to 100 mL of liquid medium and cultured at 28°C and 100 rpm for 12 h. The OD600 of Agrobacterium was adjusted to 0.6-1.0. The bacterial suspension was centrifuged at 3600 rpm and 25°C for 10 min, the supernatant was discarded, and the suspended bacterial precipitate was placed in a co-culture medium. The OD600 of Agrobacterium was adjusted to 0.8, and the suspension was inoculated into soybean Williams 82. Infected soybean cotyledonary nodes were obtained, and differentiation culture yielded clustered shoots. Resistant shoots were selected through screening culture, followed by rooting culture to obtain seedlings. After hardening off, the seedlings were transplanted and cultured again to obtain soybean T0 generation regenerated plants. A portion of leaf samples were taken, and DNA was extracted from the regenerated soybean plants using the CTAB method. Using this as a template, primer pairs were used...

[0031] SEQ ID NO.11: 5'-CCATCGTCAACCACTACATCGAGACA-3' and

[0032] SEQ ID NO.12: 5'-CTTCAGCAGGTGGGTGTAGAGCGT-3'

[0033] A 269bp Bar gene fragment was amplified, and the regenerated plants were tested to determine if they were positive. Six positive plants were obtained, which are the GmHB6 transgenic soybeans.

[0034] Wild-type soybean (Williams 82) and GmHB6 transgenic soybean were rubbed with SMV (derived from Agrobacterium containing SMV-GFP from Example 1; after inoculation of soybeans or tobacco, diseased samples were preserved as the source of SMV). Ten days later, wild-type plants showed typical mosaic symptoms, but transgenic soybeans showed milder symptoms. Figure 4 A). RT-qPCR (primer pairs SEQ ID NO.5 and SEQ ID NO.6) and western blotting results showed that the viral RNA and coat protein in soybeans overexpressing GmHB6 were significantly lower than those in the wild type. Figure 4 (BC) indicates that GmHB6 overexpression induces resistance in soybeans. In summary, these results suggest that HB6 overexpression contributes to virus resistance in plants (tobacco and soybean).

Claims

1. GmHB6 protein or the protein encoding said protein GmHB6 The application of the gene in plant antiviral activity, wherein the amino acid sequence of the GmHB6 protein is shown in SEQ ID NO.

2. GmHB6 The nucleotide sequence of the gene is shown in SEQ ID NO.1, the plant is soybean or tobacco, and the virus is soybean mosaic virus (MDV) of the genus Potatovirus Y. soybean mosaic virus ) or turnip mosaic virus ( turnip mosaic virus One or two of them.

2. A method for obtaining virus-resistant plants, characterized in that, The method includes: upregulating the expression or activity of GmHB6 protein, wherein the amino acid sequence of GmHB6 protein is shown in SEQ ID NO.2, the plant is soybean or tobacco, and the virus is soybean mosaic virus of the genus Potatovirus Y. soybean mosaic virus ) or turnip mosaic virus ( turnip mosaic virus One or two of them.

3. The method according to claim 2, characterized in that, The method for upregulating the expression or activity of GmHB6 protein is to overexpress it in the plant genome. GmHB6 Genes, the ones mentioned GmHB6 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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