CN117384941B - Gene for improving antiviral property of soybean and application thereof - Google Patents
Gene for improving antiviral property of soybean and application thereof Download PDFInfo
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Abstract
The invention discloses a gene for improving the antiviral property of soybean and application thereof, wherein the gene is named as Rmv920, the nucleotide sequence of the Rmv920 gene is shown as SEQ ID NO.1, and the application is to utilize the gene to interfere the resistance of plants to soybean mosaic virus. The invention successfully develops and confirms the efficacy of a gene named Rmv920 in resisting soybean mosaic virus and other related plant viruses; the research result of the invention has important theoretical and practical values.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a gene capable of improving the antiviral property of soybean and application thereof.
Background
Soybean is one of the main crops for food, oil and feed in China. With the increasing living standard of people, the soybean consumption demand of China is increasing. However, the soybean productivity in China can not meet domestic demands, more than 85% of the total consumption depends on import, and huge potential threat is generated to the grain safety in China. Therefore, the improvement of the soybean yield in China is helpful for relieving the contradiction between the soybean supply and demand in China. Soybean mosaic virus (Soybean mosaic virus, SMV) is a major pathogen that harms soybeans, causing serious yield loss and also seriously affecting the appearance quality of soybeans. Currently, there is no effective chemical control method, but there is a significant difference in resistance to SMV for different soybean genotypes. Therefore, breeding disease resistant varieties to reduce SMV-induced yield loss is an economical and effective control strategy. Previous research results show that soybean anti-SMV has two genetic mechanisms of anti-infection and anti-expansion. The specialization of the anti-infection varieties is strong, and the soybean production loss can be obviously reduced. But resistance is easily lost. The yield of the anti-expansion variety is slightly impaired after being infected by SMV, but the resistance is broad-spectrum and durable. In research of disease resistance genetic mechanism, research on infection resistance is concentrated at home and abroad, and research on anti-expansion is less.
Most soybean mosaic disease is a disease caused by SMV virus, and similar symptoms appear after soybean infection by virus, but the symptoms are closely related to plant genotype, pathogen and external environment. Generally, soybean mosaic symptoms can be classified into two symptoms of flowers and necrosis, wherein the flowers and leaves symptoms can be further classified into light flowers and leaves, heavy flowers and leaves, macula lutea and crinkled flowers and leaves, etc.; the symptoms of necrosis include mainly cumbrosis, vein necrosis, bud-tip or top-tip etc. (Liao Lin et al, 1990, 1992). To the extent of harm, the flower and leaf symptoms generally do not cause death of plants, but serious years cause great yield reduction; necrosis can lead to direct death of the plant, and severe years can lead to absolute production. Thus, necrosis is more damaging to soybean production.
SMV belongs to the genus Potyvirus (Potyviridae) of the family Potyviridae, the soybean mosaic virus species. SMV consists of a single-stranded sense RNA of about 9600 nucleotides (nt) in length (Hill et al, 1980), and the whole genome encodes only one open reading frame, translating from nucleotide 132 and producing 3066 ammoniaThe amino acids, transcribed to produce a polypeptide (Riechmann et al, 1992), are subsequently cleaved by three proteases (P1, HC-Pro, NIa-Pro) encoded by the viral genome to form 10 different functional mature proteins, P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and CP in sequence from 5 'to 3', where CP is the sole structural protein. In addition, a P3N-PIPO protein fused to P3 is formed inside the P3 coding region by frame-shift translation (Valli et al, 2007). The P3 protein is highly non-conserved at the 5' end as the P1 protein, and the P3 protein can affect viral accumulation and viral long distance movement within the cell, and is thought to be related to host resistance to some extent (Domier, 1987; suehiro et al, 2004). The C-terminus of P3 is associated with the expression of host symptoms and is identified as a non-toxic determinant in some Potyvirus members (Jenner et al, 2003). The P3 protein can influence genome replication and processing of multimeric proteins and is compatible withRsv1Mediated lethal systemic allergic reactions are closely related and are considered to be elicitors. However, so far as the present invention has been described,Rsv1the regulatory genes of (a) have not yet been confirmed.
In view of this, development and confirmation of genes related to soybean antiviral properties and subsequent related application studies have become a very important research topic at present.
Disclosure of Invention
The invention aims to provide a gene capable of improving the antiviral property of soybean and application thereof
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
The application of the Rmv920 gene, wherein the nucleotide sequence of the Rmv920 gene is shown as SEQ ID NO.1, is to utilize the gene to interfere the resistance of plants to soybean mosaic virus.
As a preferred embodiment of the present invention, the resistance of the target plant to soybean mosaic virus is increased by overexpressing the gene shown in SEQ ID NO.1 in the target plant.
As a preferred embodiment of the present invention, a nucleic acid sequence comprising SEQ ID NO:1, then constructing a transformant by using the overexpression vector, infecting a root system of a receptor plant by using the obtained transformant, screening positive plants, and obtaining the transgenic plant with enhanced soybean mosaic virus resistance compared with normal plants.
As a preferred technical scheme of the invention, the transformant is agrobacterium, transgenic plants are obtained by using an agrobacterium-mediated hairy root transformation method, and positive plants are screened to obtain transgenic plants with enhanced soybean mosaic virus resistance compared with normal plants.
As a preferred embodiment of the present invention, the expression of the gene is reduced or knocked out in a target plant to construct a model plant line having a higher sensitivity to soybean mosaic virus stress than a wild plant.
As a preferred technical scheme of the invention, the gene is over-expressed or reduced to express in a target plant, so that a model plant strain with different response to the soybean mosaic virus stress compared with a wild plant is constructed, and the gene is further used for identification and application development of a molecular mechanism of the plant for coping with the soybean mosaic virus stress.
The invention also includes the Rmv920 gene and its homologous or equivalent genes.
The invention also includes proteins expressed by the Rmv920 gene and its homologous or equivalent genes.
The invention also includes the use of the Rmv920 gene or its corresponding protein to increase resistance of soybean to mixed viruses; the mixed virus at least comprises soybean mosaic virus, bean common mosaic virus and watermelon mosaic virus; the resistance to the mixed virus at least comprises reduction of yellowing symptoms and top blight symptoms caused by the mixed virus.
The beneficial effects of adopting above-mentioned technical scheme to produce lie in: we have successfully developed and confirmed the efficacy of the gene designated Rmv920 in combating soybean mosaic virus and other related plant viruses; the research result of the invention has important scientific research and practical value.
Drawings
FIG. 1 is a diagram showing structural characteristics analysis of a protein expressed by the Rmv920 gene.
FIG. 2 is a graph showing changes in the expression level of Rmv920 after infection with SMV.
FIG. 3 is a schematic representation of soybean genetic transformation process (3-A) and photograph of over-expressed transgenic positive plants (3-B).
FIG. 4 is a graph showing the measurement of the target gene expression level and the virus content of the over-expressed strain.
FIG. 5 is a schematic representation of the effect of over-expressed transgenic lines on resistance to mixed viruses.
FIG. 6 is a schematic diagram showing the detection of the silencing effect of Rmv920 gene.
FIG. 7 is a schematic diagram showing the detection of the relative virus content after the silencing of the Rmv920 gene.
Detailed Description
The following examples illustrate the invention in detail. The raw materials and the equipment used by the invention are conventional commercial products, and can be directly obtained through market purchase. In the following description of embodiments, for purposes of explanation and not limitation, specific details are set forth, such as particular system architectures, techniques, etc. in order to provide a thorough understanding of the embodiments of the application. It will be apparent, however, to one skilled in the art that the present application may be practiced in other embodiments that depart from these specific details. In other instances, detailed descriptions of well-known systems, devices, circuits, and methods are omitted so as not to obscure the description of the present application with unnecessary detail. It should be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. It should also be understood that the term "and/or" as used in this specification and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations.
As used in this specification and the appended claims, the term "if" may be interpreted as "when..once" or "in response to a determination" or "in response to detection" depending on the context. Similarly, the phrase "if a determination" or "if a [ described condition or event ] is detected" may be interpreted in the context of meaning "upon determination" or "in response to determination" or "upon detection of a [ described condition or event ]" or "in response to detection of a [ described condition or event ]". In addition, in the description of the present application and the appended claims, the terms "first," "second," "third," and the like are used merely to distinguish between descriptions and are not to be construed as indicating or implying relative importance. Reference in the specification to "one embodiment" or "some embodiments" or the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," and the like in the specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless expressly specified otherwise.
Example 1
The nucleotide sequence of the Rmv920 gene is shown in SEQ ID NO.1; the amplification primer sequence comprises: rmv920F: ATGGCACTAGAATTGGTGGGTGGTGCTC (SEQ ID NO. 2); rmv920R: TTATAACAACACGTGTTCAATGCGAG (SEQ ID NO. 3). Based on the conventional knowledge of molecular biology, a nucleotide sequence having an equivalent protein expression function to the above-mentioned nucleotide sequence is also equivalent in nature to the gene of the present invention.
The paired messenger RNA and the related expressed proteins produced by gene transcription and codon-based gene expression are also one of the technical elements of the present invention.
Core protection elements of the present invention include, but are not limited to: rmv920 gene and uses thereof. Wherein the uses of the gene include: the gene is used for interfering the resistance of plants to soybean mosaic virus. This use includes at least the following several different specific application directions: (1) the resistance of the target plant to the soybean mosaic virus is improved by over-expressing the gene shown in SEQ ID NO.1 in the target plant; (2) by reducing or knocking out the expression of the gene in the target plant, the method is used for constructing a model plant strain with higher sensitivity to soybean mosaic virus stress than wild plants; (3) the gene is over-expressed or reduced to express in target plants, so that a model plant strain with different response to the soybean mosaic virus stress compared with wild plants is constructed, and the gene is further used for identification and application development of a molecular mechanism of the plants for coping with the soybean mosaic virus stress. (4) Use of the Rmv920 gene or its corresponding protein to increase resistance of soybean to mixed viruses; the mixed virus at least comprises soybean mosaic virus, bean common mosaic virus and watermelon mosaic virus; resistance to mixed viruses includes at least reduction of yellowing symptoms, top blight symptoms, etc. caused by mixed viruses.
Example 2
Subject group data modeling and deep analysis based on bioinformatics cloned from Ji Dou 12, a soybean mosaic virus-resistant variety, was namedRmv920And (3) a gene. The gene consists of 3735 nucleotides in total of 3 exons, codes for 1244 amino acids and is designated herein asRmv920. Referring to FIG. 1, analysis shows that the gene has 4 Leucine-rich repeat sequences.
Further, to clarifyRmv920The time-space expression characteristics of the genes, the expression quantity analysis of different tissues and organs of the plant shows that,Rmv920the expression quantity of the gene in the leaf and the stem is the highest, and the difference between the leaf and the stem is not obvious;Rmv920the expression quantity of the gene in flowers, seeds, roots and pods is not obvious, and the expression quantity is 24-54% of that in leaves. Further quantitatively detecting 5 time points of the infected points after the plants are infected. The results show (FIG. 2), after the plants are infected with SMV compared to CK (0 h),Rmv920there was no significant difference in the expression levels at the different time points. It is suggested that the gene may be constitutively expressed and not induced by virus.
Example 3
Cloning from Ji bean 12 using 35S as promoterRmv920Gene construction onto the overexpression vector PTF101Expression was performed in soybean susceptible variety Willmas 82, resulting in 3 overexpressing lines, see fig. 3B. Genetic transformation adopts a currently mature over-expression transgenic system; see section 3A of the drawings.
The results of detecting the target gene expression levels of 3 over-expressed strains (FIG. 4A) show that the target gene expression levels of 3 strainsRmv920The expression quantity of the gene is obviously improved compared with the control, and the expression quantity of the gene is respectively improved by 2, 14 and 11 times of the expression quantity of the control, which indicates that all 3 transgenic lines over-express the target gene. And (3) inoculating and identifying 3 over-expression strains, and detecting the soybean mosaic virus content on the upper leaves. The results show that the superexpressed strain showed a significant decrease in superfoliar Soybean Mosaic Virus (SMV) content compared to the Wild Type (WT), with #oe_rmv920_1 virus concentration around 38.6% of WT, and #oe_rmv920_2 and #oe_rmv920_3 virus concentrations of only 8.6% and 8.9% of WT virus concentration. The result shows that after the gene Rmv920 is over-expressed, the concentration of SMV virus can be obviously inhibited. The method provides reliable resistance gene resources for subsequent disease-resistant breeding work,
example 4
In addition to the soybean mosaic virus resistance produced by overexpression, we have unexpectedly found that overexpressed transgenic plants also have resistance to mixed viruses. In view of the complex types of viruses in field production, in order to further analyze whether the gene can have an effect on mixed infection of multiple viruses, a mixed virus following method is adopted for resistance test. The mixed virus is obtained by directly sampling in the field, and is detected by a PCR or ELISA (enzyme linked immunosorbent assay) method to prove that the mixed virus contains a plurality of plant viruses such as soybean mosaic virus, bean common mosaic virus, watermelon mosaic virus and the like. The results of the artificial inoculation identification show (FIG. 5) that typical virus symptoms such as obvious yellowing, top necrosis and the like appear after wild type inoculation, and in the 3 over-expression strains, no obvious virus symptoms such as necrosis or top blight and the like appear in the # OE_Rmv920_2 and the # OE_Rmv920_3 except a small number of necrotic spots appear in the # OE_Rmv920_1. In addition, the degree of yellowing of #oe_rmv920_1, #oe_rmv920_2, and #oe_rmv920_3 in the 3 over-expressed lines was sequentially reduced. The experimental results show that the expression of Rmv920 can obviously reduce the yellowing and necrosis symptoms caused by mixed viruses, and has obvious promotion effect on soybean disease resistance.
Example 5
And designing a virus-induced gene silencing (Virus induce gene silence, VIGS) silencing primer according to the conserved region, and identifying whether the primer has the effect of inhibiting virus propagation or infection by reducing the expression quantity of the target gene. For the purpose ofRmv920The gene design silencing primer has the following sequence:
For: AACACCCAAAGGATCCGGTGGTGCTCTTCTTTCTGCTT（SEQ ID NO.4）；
Rev: CTCGTTATATTGTGGCCAACTAACAGGAGAAGATTTGAAG（SEQ ID NO.5）；
after in vitro transcription, the soybean infectious disease variety Nannong 1138-2 is infected, the leaves with typical mosaic virus symptoms are collected, and the leaves are frozen by liquid nitrogen and stored in a refrigerator at-80 ℃ for standby. In addition, RNA of soybean leaves with typical symptoms is extracted, and after inversion, gene silencing effect is detected by using the following primers, for ATCTACAGCCTTCCAAACAC; rev CCCATTCTTCCCATTCCTTC. Results show (FIG. 6), post-silencing plantsRmv920The expression level of the vector is about 40% of that of the control, which indicates that the constructed vector can effectively silenceRmv920Is expressed by (a).
Furthermore, successful in vitro transcribed VIGS silencing isolates silence the material Ji bean 12 carrying candidate disease resistance genes and carry out infection identification by utilizing recombinant SMV strain HB-RS. Fluorescent quantitative primers were designed for HB-RS, specific primer sequences were SF CCGATGGTTGAAGGGAAGAT and SR ACACCCATCTGCTCATCATC, virus concentration was calculated by 2-DeltaCT method, and internal reference gene was EF-1α (TefS 1; NCBI accession number: X56856). Because of BPMV mediated gene silencing, two cases of BPMV and SMV complex infection and BPMV single infection may occur, in order to further detect whether the genes are directly related to SMV infection, qRT-PCR was used to detect leaf blades of the plant infected with SMV after silencing, and after Ji beans 12 are inoculated with SMV, bpmv+smv and sirmv920+smv, the results show (fig. 7) that the virus content difference between the single infection of SMV and the co-infection of BPMV and SMV is not significant, indicating that the co-infection of BPMV and SMV does not cause the increase of the SMV virus content. In addition, the results also showed silencingRmv920post-SMV infection plant concentrationRaised by 440%, proveRmv920Has remarkable effect of inhibiting viruses.
In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.
The above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
Claims (1)
- Use of an Rmv920 gene to increase resistance of soybean to mixed viruses, wherein the nucleotide sequence of the Rmv920 gene is shown in SEQ ID No.1; the method is characterized in that: the mixed virus at least comprises soybean mosaic virus, bean common mosaic virus and watermelon mosaic virus; the resistance to the mixed virus at least comprises reduction of yellowing symptoms and top blight symptoms caused by the mixed virus.
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