Soybean Mosaic Virus Resistance Gene GmMATE and Its Application

Abstract

The present invention discloses a soybean mosaic virus resistance gene, GmMATE, and its application. The nucleotide sequence of the gene is shown in SEQ ID NO. 1. The application is to utilize the gene to intervene in plant resistance to soybean mosaic virus. The present invention has important significance in theoretical research and agricultural and forestry applications, especially crop breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, in particular to a gene capable of improving the antiviral performance of soybean and application thereof. Background Art

[0002] Plants develop a variety of defense mechanisms to cope with infection by pathogens. These defenses can be categorized into two main groups based on the host's response to infection: resistance to infection (vertical resistance) and resistance to spread (horizontal resistance). These two defenses are distinctly different. Resistance to infection confines the pathogen to the point of infection, preventing systemic spread. Resistance to spread, on the other hand, allows the virus to complete systemic infection but limits viral load to a low level, minimizing the risk to plant growth and development. Alternatively, the plant can compensate for the damage caused by the virus to a certain extent by adjusting its own metabolic mechanisms, thereby reducing the adverse effects on plant growth, reproduction, and yield (Martin et al. 2003; Salomon 1999).

[0003] Soybean mosaic disease (SMV) is a major pest of soybeans, occurring worldwide. Soybean mosaic virus (SMV), the primary pathogen causing soybean mosaic disease, can lead to reduced soybean yield, decreased quality, and even crop failure. Currently, there are no effective chemical control methods for viral diseases caused by SMV in soybeans, primarily focusing on prevention and control. Therefore, breeding resistant varieties has become the most cost-effective approach. To mitigate the impact of SMV on soybean production, breeders use artificial inoculation to identify resistance in varieties to be used for production and promotion. The virus strains used are generally locally prevalent. Varieties are also graded using the disease index (DI) (Bai Li et al., 2007; Zhi Haijian et al., 2005; Li Kai et al., 2013). However, this artificial inoculation method is labor-intensive and occurs late in the breeding process. Once susceptible varieties are identified, all previous work is lost. Molecular marker-assisted breeding is currently considered a breeding strategy with broad application prospects (He et al. 2014; Ottoman et al. 2009; Fan et al. 2004). However, its application requires the discovery and identification of genes with important breeding value. Consequently, researchers both domestically and internationally have conducted extensive research on SMV resistance genes. For example, using G lines, researchers abroad have identified three reliable loci: Rsv1, Rsv3, and Rsv4 (Yu et al. 1994; Jeong et al. 2002, Jeong and Maroof, 2004; Maroof et al. 2010). Based on the gene-for-gene theory (Flor, 1971), domestic researchers have also identified more than ten resistance genes in soybeans that are specific to SC lines (Wang et al. 2011; Wang Dagang et al. 2014; Yang et al. 2010, 2013; Zheng et al. 2014).

[0004] Necrosis is a common symptom of soybean mosaic disease. Although the hypersensitive response (HR) is generally considered a plant response associated with disease resistance, some examples suggest that HR is unrelated to disease resistance and that HR symptoms can be induced by a variety of pathways (Morel et al. 1997). Furthermore, although the specific molecular mechanisms and effects of HR remain unclear, it is known that some stress defense responses produce secondary metabolites that are harmful to plant cells, including reactive oxygen intermediates, phytoalexins, and salicylic acid (Ward et al. 1991). When these harmful metabolites accumulate to a certain level, they cause cell death, resulting in HR or HR-like reactions. Therefore, eliminating these harmful toxins or substances from cells and maintaining them within a tolerable dose is crucial for plant life (Hill et al. 2011). The MATE transporter family is a class of genes involved in the efflux of toxins and other metabolites (He et al. 2010). The MATE (Multidrug and Toxic Compound Extrusion Transporter) family of genes encodes citrate transporters that respond to a variety of biotic and abiotic stresses, including aluminum stress and phosphorus and iron deficiencies (Santos et al. 2017; Zhou et al. 2014; Maron et al. 2013; Qiu et al. 2017). In Arabidopsis, overexpression of MATE family genes from rice not only altered plant growth rate and morphology but also increased plant sensitivity to biotic and abiotic stresses, with a significant reduction in the expression of defense-related genes (Tiwari et al. 2012). Studies have also shown that MATE transporters can protect cells from oxidative stress, a condition highly correlated with plant disease symptoms. However, the biological role of MATE family genes in soybean mosaic disease resistance has yet to be reported.

[0005] In summary, the research on genes related to soybean antiviral properties and the development of subsequent related applications are a technical hotspot and difficulty that urgently needs to be addressed. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a gene capable of improving the antiviral performance of soybean and its application.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] The present invention comprises a soybean mosaic virus resistance gene, and its nucleotide sequence is shown in SEQ ID NO.1 (sequence table in XML format).

[0009] The present invention also includes homologous genes of the soybean mosaic virus resistance gene or known or unknown genes with biological equivalence.

[0010] The present invention also includes RNA corresponding to the above-mentioned genes, including messenger RNA and / or other long-chain or short-chain RNA and / or functional RNA.

[0011] The present invention also includes the protein or polypeptide expressed by the above gene.

[0012] The present invention also includes the use of the above gene in improving or reducing the resistance of target plants to soybean mosaic virus.

[0013] As a preferred technical solution of the present invention, the target plant is overexpressed with the target gene shown in SEQ ID NO.1 to improve the resistance of the target plant to soybean mosaic virus.

[0014] As a preferred technical solution of the present invention, an overexpression vector containing the nucleotide sequence shown in SEQ ID NO: 1 is first constructed, and then a transformant is constructed using the overexpression vector. The resulting transformant is then used to infect the root system of a recipient plant, and positive plants are screened to obtain transgenic plants with enhanced soybean mosaic virus resistance compared to normal plants.

[0015] As a preferred technical solution of the present invention, specifically, 35S is used as a promoter, the CDS sequence of the target gene is constructed into the overexpression vector PTF101, the resulting recombinant expression vector is expressed in the disease-susceptible soybean variety Nannong 1138-2, and positive plants are screened to obtain transgenic plants with enhanced soybean mosaic virus resistance compared with normal plants.

[0016] The present invention also includes a recombinant expression vector, comprising a backbone vector and the gene according to claim 1 or 2 or its equivalent nucleotide sequence.

[0017] The present invention also includes engineered bacteria, transformants or other intermediates of molecular biological operations containing the above-mentioned genes or recombinant expression vectors.

[0018] The beneficial effect of the above-mentioned technical solution lies in the development and confirmation of the efficacy of a gene, designated GmMATE, in resisting soybean mosaic virus. Previous research, through QTL mapping, identified a QTL locus that influences necrosis symptoms after mosaic virus infection. This study, through further in-depth research using family analysis, gene expression, and transgenic methods, ultimately confirmed that GmMATE (gene ID: Glyma.03G005600) is a key regulatory gene, possessing significant scientific and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 shows the tissue expression analysis of 6 MATE candidate genes.

[0020] Figure 2 This is the experimental map for the correlation analysis between the qTsmv-3 site and the salicylic acid resistance pathway.

[0021] Figure 3 This is the experimental map of Glyma.03G005600 transformation into Arabidopsis thaliana.

[0022] Figure 4 This is a detection picture of a transgenic positive soybean plant.

[0023] Figure 5 Schematic diagram of the main nodes of soybean genetic transformation technology operations.

[0024] Figure 6 This is a detection chart of target gene expression and virus content in overexpression strains in soybean. DETAILED DESCRIPTION

[0025] The present invention is described in detail in the following examples. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly purchased from the market.

[0026] It should be understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0027] As used in this specification and the appended claims, the term "if" can be interpreted as "when" or "upon" or "in response to determining" or "in response to detecting," depending on the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "upon determination" or "in response to determining" or "upon detection of [described condition or event]" or "in response to detecting [described condition or event]," depending on the context.

[0028] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0029] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in yet other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized.

[0030] The terms "include", "comprising", "having" and variations thereof mean "including but not limited to", unless specifically emphasized otherwise.

[0031] Example 1. Analysis of expression characteristics of candidate MATE genes at the qTsmv-3 locus

[0032] In our previous research, we identified a QTL locus qTsmv-3 that affects the necrosis symptoms caused by mosaic virus infection through QTL positioning.

[0033] On this basis, this study finally confirmed that GmMATE (gene number: Glyma.03G005600) is its key regulatory gene through family analysis, gene expression, transgenic and other means.

[0034] First, to clarify the evolutionary characteristics of MATE family genes in the soybean genome, we predicted 128 MATE family genes from the public genome Williams82v2.1 (https: / / phytozome-next.jgi.doe.gov / ) and downloaded their corresponding protein sequences. Phylogenetic analysis was then performed with 12 MATE family genes reported in Arabidopsis, rice, rapeseed, tobacco, barley, and maize. The results showed that the 128 soybean MATE family genes could be clearly divided into five subfamilies.

[0035] The six MATE candidate genes (CG1-CG6) within the qTsmv-3 locus were predicted on a public database website (https: / / soyatlas.venanciogroup.uenf.br). The expression results based on 4124 samples showed that the six candidate genes showed certain differences in tissue expression. Figure 1Analyzing expression levels, CG5 showed generally higher expression in leaves and stems aboveground, while CG6 showed higher expression in roots and nodules belowground. Furthermore, analysis of the primary expression sites revealed that CG1, CG2, CG4, and CG5 were similar, primarily expressed aboveground, with relatively low or almost no expression in roots. CG3 was significantly expressed both aboveground and belowground. These results indicate that the primary organs in which the six MATEs function differ, suggesting that some genes may also have distinct biological functions.

[0036] Example 2. Correlation analysis between the qTsmv-3 locus and the salicylic acid resistance pathway

[0037] To further analyze whether the regulation of SMV necrosis symptoms by the qTsmv-3 locus is related to the salicylic acid (SA) resistance pathway, the expression levels of the indicator genes ICS and PR1 in the SA resistance pathway were analyzed after inoculation with the SMV strain SC3 using the near-isogenic lines of the qTsmv-3 locus as materials. The results showed that ( Figure 2 A), the symptoms of the near-isogenic lines showed obvious differences 25 days after inoculation. No obvious necrotic symptoms were observed in the inoculated leaves and upper leaves of #NIL-SMC. Obvious mosaic symptoms were observed 7 days after inoculation. 25 days after inoculation, the upper leaves showed obvious mosaic and wrinkling, two typical disease symptoms. In contrast, #NIL-NC showed mild mosaic symptoms 7 days after inoculation, but then mesophyll necrotic spots appeared, and some necrosis occurred along the veins. This is basically consistent with our previous observations (Lin et al., 2021). Expression analysis results showed that after inoculation, the expression levels of ICS and PR1 in #NIL-NC were upregulated by 2.40 and 15.16 times, respectively, compared with #NIL-SMC ( Figure 2 B, C), indicating that there are significant differences in the SA resistance process between #NIL-NC and #NIL-SMC. In addition, the concentration of SC3 in #NIL-NC leaves is about 30% of that in #NIL-SMC, indicating that the differences in the SA resistance process between near-isogenic lines affect the virus content in leaves ( Figure 2 D) The above results indicate that the qTsmv-3 locus is involved in SA-mediated resistance to SMV.

[0038] Example 3. Experimental verification in model plants

[0039] In order to further verify the biological function of Glyma.03G005600 and whether it is involved in the SA disease resistance process, Glyma.03G005600 was transformed into Arabidopsis thaliana (Genetic Resource Source Disclosure Registration Form) for observation.

[0040] The results showed that the leaf number, leaf size, root size and total biomass of the transgenic plants (OE_MATE) were significantly greater than those of the wild type (WT) ( Figure 3A, B), indicating that Glyma.03G005600 can promote plant growth and development. In addition, about 7 days after ultraviolet (UV-B) treatment, the leaves and stems of transgenic Arabidopsis plants showed obvious purple coloration, indicating that anthocyanins had accumulated significantly, while the wild type (WT) did not show such a phenomenon. Further fluorescence quantitative detection of the indicator genes ICS1 and PR1 on the SA pathway at this stage showed that compared with plants not treated with UV-B, ICS1 and PR1 were significantly upregulated in both transgenic and wild-type plants after UV-B treatment ( Figure 3 D, E). In the UV-B treatment group, the expression levels of ICS1 and PR1 in OE_MATE were significantly increased by 4.22-fold and 9.12-fold compared with WT, respectively ( Figure 3 D, E), indicating that Glyma.03G005600 can significantly promote the synthesis of SA and the stress response process of the SA pathway.

[0041] Example 4. Verification of overexpression function in soybean

[0042] To further confirm the biological function of Glyma.03G005600 in SMV resistance, the CDS sequence of Glyma.03G005600 cloned from Jidou 12 (Genetic Resource Source Disclosure Registration Form) was constructed into the overexpression vector PTF101 using 35S as the promoter. GmMATE was expressed in the susceptible soybean variety Nannong 1138-2 (Genetic Resource Source Disclosure Registration Form), and three overexpression lines were obtained ( Figure 4 Among them, the main nodes of soybean genetic transformation technology operation include (see Appendix Figure 5 ):

[0043] Preparation of infection solution: prepare explants and bacterial solution to infect explants (3 hours), incubate at a constant temperature of 22 degrees (3 days), bud recovery medium (7 days), bud induction medium (21 days), and bud elongation medium.

[0044] Soybean seed sterilization: Before sterilization, select beans that are large, plump, smooth, without holes, wrinkles, or cracks. Then, spread six culture plates evenly. Place a large desiccator in a fume hood. Place an uncovered blue-mouthed bottle in the middle of the desiccator. Place the culture plates containing the beans next to them, lift the lid, and place it against the desiccator. Pour 90 mL of NACLO, followed by 10 mL of concentrated HCl, into the blue-mouthed bottle and quickly close the lid. Sterilize for 15-16 hours.

[0045] Prepare the infection solution: Prepare activated Agrobacterium by adding approximately 25-30 ml of infection solution (100 ml) to a 50 ml centrifuge tube. Use a wire loop to scrape some of the culture solution from the culture dish. Add this to the bottle containing the infection solution, shake well, and measure the OD600 value. Adjust the final OD value to 0.6. After adjusting the OD value, evenly distribute the solution into two clean, sterile culture dishes, 50 ml each, and set aside.

[0046] Soybean explant preparation: Cut soybean seeds, germinated overnight, in half in a laminar flow hood, retaining the cotyledons, hypocotyl, and plumule. True leaves can also be removed to increase the wound opening. Place the cut beans in the infiltrate dye solution and let them sit for 3 hours.

[0047] Incubate at a constant temperature of 22 degrees: Drain the beans, then place them in a new culture dish with the hypocotyl side facing up. Place 15 petals in one dish and place it in a constant temperature incubator at 22 degrees for 3 days.

[0048] Bud Recovery: Remove the cotyledonary nodes after incubation and observe for bacterial growth. Use forceps to pick up the sterile explant bean petal, with the embryonic axis facing downward. Insert the petal vertically into the R0 medium, ideally with both flat surfaces facing in the same direction. Seal the tube and incubate in a light chamber for 7 days.

[0049] Bud induction: Take out the cotyledonary node with buds from the R0 culture medium. At this time, the hypocotyl and buds are elongated, and some have long roots. Place them on the lid of the culture dish, cut off the roots and cotyledons, and press the part above the growth point node into the R1 culture medium, which contains the selection agent glufosinate. Culture in the photoculture chamber for 21 days.

[0050] Bud elongation stage: After screening in the bud induction stage, successfully transformed plants will show Basta resistance. The surrounding aging and dead adventitious buds and leaves are cleaned and transferred to bud elongation medium and cultured in a light culture chamber for 21 days.

[0051] Obtaining positive plants: The plants obtained at the bud elongation stage are tested for Basta resistance. The positive plants are transferred to rooting medium and cultured until they take root. After taking root, they are transplanted.

[0052] The results of the detection of target gene expression levels in the three overexpression lines showed that ( Figure 6A), the expression levels of GmMATE in the three strains were significantly higher than those in the control, increasing by 11.37, 16.76, and 12.98 times the expression levels in the control, respectively, indicating that the target gene was overexpressed in all three transgenic strains. After further inoculation and identification of the three overexpression strains, the soybean mosaic virus content was detected in the upper leaves. The results showed that compared with the wild type (WT), the soybean mosaic virus (SMV) content in the upper leaves of the overexpression strains showed a significant decrease, among which the virus concentration of #OE_1 was about 23.43% of that of WT, and the virus concentrations of #OE_2 and #OE_3 were 31.141% and 54.41% of that of WT. The results show that after overexpressing the gene GmMATE, the concentration of SMV virus can be significantly inhibited. This provides a reliable resistance gene resource for subsequent disease resistance breeding.

[0053] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0054] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention.

Claims

1. Use of a gene for improving soybean resistance to soybean mosaic virus; wherein: The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The target gene shown in SEQ ID NO. 1 is overexpressed in the target soybean to improve the resistance of the target soybean to soybean mosaic virus.

3. The use according to claim 2, characterized in that: First, an overexpression vector containing the nucleotide sequence shown in SEQ ID NO: 1 is constructed, and then a transformant is constructed using the overexpression vector. The resulting transformant is then used to infect the root system of a recipient plant, and positive plants are screened to obtain transgenic plants with enhanced soybean mosaic virus resistance compared to normal plants; the plant is soybean.

4. The use according to claim 3, characterized in that: Specifically, using 35S as a promoter, the CDS sequence of the target gene was constructed into the overexpression vector PTF101, and the resulting recombinant expression vector was expressed in the susceptible soybean variety Nannong 1138-2. Positive plants were screened to obtain transgenic plants with enhanced soybean mosaic virus resistance compared to normal plants; the plant was soybean.

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