Gspp13l gene, recombinant vector thereof, recombinant bacteria and application thereof

Abstract

GsRPP13L The gene, its recombinant vector, recombinant bacteria and application belong to the technical field of plant disease resistance. In order to solve the technical problems that the utilization of SMV resistance genes and the analysis of disease resistance mechanism are not comprehensive enough in the prior art, a broad-spectrum and durable SMV-resistant soybean variety cannot be quickly and efficiently bred, and the resistance variety is prone to "resistance failure", a soybean gene for improving resistance to soybean mosaic virus is provided GsRPP13L The gene is shown in the nucleotide sequence SEQ ID NO.1, and the gene can improve the resistance of tobacco or soybean to soybean mosaic virus through experiments, and the present application provides a theoretical basis for plant resistance to soybean mosaic virus.

Description

Technical Field

[0001] This invention belongs to the field of plant disease resistance technology, specifically involving GsRPP13L Genes, their recombinant vectors, recombinant bacteria, and their applications. Background Technology

[0002] Soybean Mosaic Virus (SMV) is the most widespread and damaging viral disease of soybeans globally. SMV is transmitted by non-persistent vectors such as aphids and can infect soybeans throughout their entire growth cycle. Infected plants exhibit mosaic, wrinkled, and deformed leaves, stunted stems, a sharp decrease in pod setting, shriveled grains, and reduced protein content. In severe cases, yield losses can reach 30%-80%, or even total crop failure. Adding to the challenge is the existence of over 20 distinct pathogenic strains of SMV, including SC, SG, and Sh. The viral genome is prone to mutation, and single resistance genes are easily overcome, leading to "resistance failure" in resistant varieties and exacerbating the risk of disease outbreaks.

[0003] Currently, accurate identification of SMV resistance genes and germplasm resources is key to solving the above problems. On the one hand, marker-assisted selection (MAS) or gene editing technology can be used to directionally aggregate resistance genes from different strains (such as...). Rsv1 , Rsv3 , Rsv4 (etc.) to create new soybean varieties with broad-spectrum and long-lasting disease resistance, avoiding the blindness and inefficiency of "screening based on phenotype" in traditional breeding; on the other hand, analyzing the mechanism of action of resistance genes can provide theoretical support for antiviral molecular design breeding.

[0004] However, the utilization of SMV-resistant genes and the comprehensive analysis of disease resistance mechanisms in existing technologies still need improvement, and cannot fully meet the demand for rapid and efficient breeding of broad-spectrum SMV-resistant soybean varieties. Summary of the Invention

[0005] To address the shortcomings in existing technologies regarding the utilization of SMV resistance genes and the analysis of disease resistance mechanisms, which hinder the rapid and efficient breeding of broad-spectrum, durable SMV-resistant soybean varieties and lead to "resistance failure" in resistant varieties, this invention provides the following technical solution: The first objective of this invention is to provide a soybean for improving resistance to soybean mosaic virus. GsRPP13L Genes, the soybean GsRPP13L The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0006] The second object of the present invention is to provide the above-mentioned soybeans. GsRPP13LApplication of genes in enhancing resistance of tobacco or soybean to soybean mosaic virus.

[0007] In one embodiment of the invention, the application is achieved by overexpressing soybean in tobacco. GsRPP13L Genes that enhance tobacco resistance to soybean mosaic virus, or overexpress soybean in soybeans. GsRPP13L Genes enhance soybean resistance to soybean mosaic virus.

[0008] The third object of the present invention is to provide a product containing the above-mentioned soybeans. GsRPP13L Gene recombination vectors.

[0009] In one embodiment of the present invention, the carrier skeleton of the recombinant vector is pCB301 or pCAMBIA3301.

[0010] A fourth objective of this invention is to provide the application of the above-mentioned recombinant vector in improving the resistance of tobacco or soybean to soybean mosaic virus.

[0011] The fifth object of the present invention is to provide a product containing the above-mentioned soybeans. GsRPP13L Recombinant bacteria.

[0012] In one embodiment of the present invention, the starting strain of the recombinant bacteria is Agrobacterium.

[0013] Further specifying, the Agrobacterium is Agrobacterium EHA105 or Agrobacterium GV3101.

[0014] A sixth objective of this invention is to provide the application of the above-mentioned recombinant bacteria in improving the resistance of tobacco or soybean to soybean mosaic virus.

[0015] Beneficial effects: This invention achieves transient overexpression in tobacco GsRPP13L Genes, transient overexpression discovered GsRPP13L In tobacco leaves treated with the gene, the intensity of the GFP fluorescence signal was significantly weaker than that in the negative control group and other candidate gene treatment groups, proving that... GsRPP13L Gene expression effectively inhibited the replication and proliferation of SMV in Nicotiana benthamiana. Furthermore, transient overexpression was detected by qRT-PCR. GsRPP13L The relative expression levels of SMV genomic RNA in tobacco leaves and negative control leaves were compared, and it was found that overexpression was observed in the tobacco leaf leaf compared to the negative control. GsRPP13L The relative expression level of SMV genomic RNA was significantly reduced in tobacco leaves, further validating transient overexpression. GsRPP13L The gene can significantly inhibit the proliferation of SMV in Nicotiana benthamiana.

[0016] This invention overexpresses in soybeans GsRPP13L Genes, overexpression discovered GsRPP13L The relative expression level of SMV genomic RNA in the inoculated leaves of transgenic soybean plants was reduced by approximately 50%-70% compared to wild-type, indicating overexpression. GsRPP13L The relative expression level of SMV genomic RNA in the phylogenetic leaves of transgenic soybean plants was reduced by approximately 80%-90% compared to the wild type. This result indicates that the disease resistance gene... GsRPP13L It is expressed not only in the leaves of transgenic plants but also in phloem cells, resulting in transgenic plants inhibiting the virus not only in the first stage (initial infection of the virus) and the second stage (intercellular movement of the virus) but also in the third stage of viral infection (systemic movement of the virus). Attached Figure Description

[0017] Figure 1 for GsRPP13L Image showing the results of agarose gel electrophoresis identification of gene amplification; Figure 2 This is a schematic diagram illustrating the construction of the transient overexpression vector pCB301:GsRPP13L-MYC; Figure 3 The image shows the agarose gel electrophoresis results of the transient overexpression vector pCB301:GsRPP13L-MYC; where EV represents the empty vector. Figure 4 This is a schematic diagram of the structure of the SMV-GFP infectious cloning vector carrying a GFP marker; Figure 5 The graph shows the inhibitory effect of transient overexpression of four candidate genes on SMV in tobacco leaves; where A represents transient overexpression. Glyma.13G184800 The inhibitory effect of the gene on SMV in tobacco leaves, B represents transient overexpression. Glyma.13G184900 The inhibitory effect of the gene on SMV in tobacco leaves, where C represents transient overexpression. Glyma.13G187900 The inhibitory effect of the gene on SMV in tobacco leaves, where D represents transient overexpression. Gs.13GNEW1 The inhibitory effect of the gene on SMV in tobacco leaves; Figure 6 For transient overexpression GsRPP13L The inhibitory effect of tobacco leaves on SMV is shown in the figure; where A is the fluorescence result and B is the result of the detection of the relative expression level of SMV genomic RNA. Figure 7 A schematic diagram illustrating the construction of the overexpression vector pCAMBIA3301-GsRPP13L; Figure 8 The image shows the agarose gel electrophoresis results of the overexpression vector pCAMBIA3301-GsRPP13L; where EV represents the empty vector. Figure 9 Four plants GsRPP13L Image showing the identification results of soybean plants with overexpressing genes; Figure 10 For overexpression GsRPP13L The inhibition effect of the gene on SMV in soybean leaves is shown in the figure. Among them, A is the fluorescence image of the inoculated leaves, B is the detection result of the relative expression level of SMV genomic RNA in the inoculated leaves, C is the fluorescence image of the system leaves, and D is the detection result of the relative expression level of SMV genomic RNA in the system leaves. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0020] The SMV infectious cloning vector pSMV-GFP carrying the GFP label and the recombinant soybean mosaic virus SMV-GFP expressing green fluorescent protein used in the following examples are both disclosed in the following literature: Zhou et al. Genome-wide association study of soybean mosaic virus resistance with a GFP-based rapidevaluation system. Agronomy. 2025, 15: 1960. Example 1: A GsRPP13L Gene acquisition and its application in improving tobacco resistance to soybean mosaic virus. 1. Discovery of parental materials—Resistance identification of wild soybean whole-genome introduction lines Using wild soybean ZYD00006 as the donor parent and cultivated soybean Suinong 14 (SN14) as the recipient parent, a chromosome segment introduction line population containing 192 lines was constructed through years of continuous hybridization, backcrossing, and marker-assisted selection. This introduction line population covers the entire genome of wild soybean ZYD00006. Since 2021, the resistance of the introduction line population to SMV has been identified through field and pot artificial friction inoculation experiments for several consecutive years. It was found that 9 of the 192 introduction lines showed high resistance to SMV. One of the lines (numbered R172) had a stable SMV resistance phenotype and good other agronomic traits. Subsequently, R172 was used as the disease-resistant parent for disease resistance gene mining and was named disease-resistant parent R172.

[0021] 2. Location of disease-resistant genes The resistant parent R172 was crossed with the susceptible parent SN14, and the F1 generation was then self-crossed to obtain the F2 generation. All F1 generations showed resistance, while the F2 generation showed a 3:1 segregation ratio of resistant to susceptible. This result indicates that the resistance of the resistant parent R172 to SMV is controlled by a pair of major genes.

[0022] In the F2 segregating population, 20 resistant plants and 20 susceptible plants with distinct resistance and susceptibility phenotypes were selected to construct resistance and susceptibility pools, respectively. Whole-genome resequencing pooled association analysis (BSA) was then performed. A significantly correlated region of approximately 4-5 Mb was located on soybean chromosome 13. Alignment with the soybean reference genome Williams82_a4_v1 revealed approximately 500 known genes within this region.

[0023] 3. Identification of candidate genes Considering that the disease resistance phenotype of parental R172 conforms to the expression of disease resistance response mediated by immune receptor-like disease resistance proteins (NLR proteins), in order to further narrow down the candidate gene range, bioinformatics methods were used to screen NLR proteins (CNL and TNL classes) within the above-mentioned segments by comparing CC / TIR, NB-ARC, and LRR domains. The NLR proteins within these segments were predicted, and a total of 16 NLR-like disease resistance proteins were identified. Based on the whole genome sequences of SN14 and wild soybean ZYD00006 already determined by our research group, 5 of the above 16 NLR proteins in ZYD00006 were completely identical to SN14. The gene sequences of the remaining 11 NLR proteins were analyzed in R172, and the results showed that R172 carries 5 NLR genes from the disease-resistant donor parental ZYD00006. Therefore, these 5 NLR genes were selected as candidate genes. Glyma.13G184800 , Glyma.13G184900 , Glyma.13G187900 , Gs.13GNEW1 and Glyma.13G190800 ( GsRPP13L ).

[0024] 4. Functional verification of candidate genes (1) Cloning of candidate genes Based on the whole genome sequence of wild soybean ZYD00006, specific primers were designed at both ends of the CDS sequences of five candidate genes, and primers were synthesized by a biotechnology company, resulting in five primer pairs for cloning the candidate genes. Among them, the primers used for cloning... GsRPP13L The primer pairs for the genes consisted of RPP13L-F (nucleotide sequence as shown in SEQ ID NO.3: 5'-ATGGCACTAGAATTGGTTGGTG-3') and RPP13L-R (nucleotide sequence as shown in SEQ ID NO.4: 5'-CTAGAGCAACGGACAATCGAG-3'). Leaves of wild soybean ZYD00006, 6 h after artificial inoculation with SMV, were used. Total RNA was extracted from the leaf tissue and reverse transcribed to obtain cDNA. Using the cDNA as a template, PCR amplification was performed on the five candidate genes using the aforementioned five primer pairs to obtain the CDS sequences of the five candidate genes. GsRPP13L The PCR products of the gene were identified by agarose gel electrophoresis, and the results are shown in [the table below]. Figure 1 ,Depend on Figure 1 It can be seen that, GsRPP13L The CDS sequence fragment size of the gene was as expected. The target band separated by agarose gel electrophoresis was purified using a gel extraction kit and then sent to a sequencing company for sequencing. The sequencing results showed... GsRPP13L The gene's CDS sequence is 3591 bp (nucleotide sequence as shown in SEQ ID NO.1), encoding 1196 amino acids (amino acid sequence as shown in SEQ ID NO.2). GsRPP13L The CDS sequence of the gene was analyzed by BLASTn in the GenBank database. The results showed that it had the highest sequence identity (95.14%) with wild soybean RPP13-like 1 protein mRNA (XR_001388786.3) and soybean RGA4 protein mRNA (XM_028381977.1), with a coverage of 97% for both.

[0025] (2) Construction of plant transient overexpression vectors containing candidate genes The CDS sequences of the five candidate genes obtained above were cloned downstream of the 35S promoter of the plant expression vector pCB301 (nucleotide sequence shown in SEQ ID NO. 9) to construct transient overexpression vectors pCB301:Glyma.13G184800-MYC, pCB301:Glyma.13G184900-MYC, pCB301:Glyma.13G187900-MYC, pCB301:Gs.13GNEW1-MYC, and pCB301:GsRPP13L-MYC (the transient overexpression vectors were constructed and validated by Weimi Biotechnology Co., Ltd., and the provided product is the final product after successful sequencing). The construction diagram of the transient overexpression vector pCB301:GsRPP13L-MYC is shown below. Figure 2 As shown in the figure, the agarose gel electrophoresis identification results are as follows. Figure 3 As shown, the primer pair used for identification consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.5 (5'-TTCTCAAGGAAAATCAACTGAA-3') and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.6 (5'-CATCACTAATTGGATTCATTTC-3').

[0026] (3) Screening for host genes that inhibit SMV proliferation using Agrobacterium tumefaciens infiltration inoculation method ① Preparation of experimental materials Plant material: Healthy Tobacco Benedict ( Nicotiana benthamiana ) plant.

[0027] Strains and vectors: Agrobacterium GV3101 was transformed into the following plasmids: Experimental group: transient overexpression vectors carrying 5 different candidate genes (pCB301:Glyma.13G184800-MYC, pCB301:Glyma.13G184900-MYC, pCB301:Glyma.13G187900-MYC, pCB301:Gs.13GNEW1-MYC, pCB301:GsRPP13L-MYC); Virome: SMV infectious cloning vector pSMV-GFP carrying GFP label (the structure of vector pSMV-GFP is shown in Figure 1) Figure 4 (as shown) Control group: control plasmid pCB301:MYC carrying the empty vector.

[0028] ② Preparation of Agrobacterium-containing liquid Activation: Take 10 μL of Agrobacterium GV3101 bacterial suspension stored at -80°C, inoculate it into 1 mL of liquid LB medium containing the corresponding antibiotic, and culture at 28°C and 250 rpm for 24-36 hours until the bacterial suspension becomes turbid to obtain activated bacterial suspension.

[0029] Expanded culture: Take 500 μL of the above activated bacterial solution and inoculate it into 25 mL of fresh liquid LB medium (supplemented with 50 μg / mL kanamycin, 10 mmol / L 2-(N-morpholine) ethanesulfonic acid, pH=6.2, and 20 μmol / L acetylsyl syringone), and continue to culture for about 12 hours under the same conditions.

[0030] Cell collection and resuspending: Collect cells by centrifugation at 5000×g for 6 minutes at 4°C, discarding the supernatant; gently resuspend the cells in inoculation buffer (10 mmol / L magnesium chloride, 10 mmol / L 2-(N-morpholine)ethanesulfonic acid, pH=6.2, 100 μmol / L acetylsylgenone); adjust the bacterial concentration with inoculation buffer to achieve the desired OD value. 600 When the value reaches 0.6-0.8, the inoculation working solution is obtained.

[0031] ③ Mixed inoculation and plant treatment Inoculum mixing: Agrobacterium inoculation solution carrying the candidate gene was mixed with pSMV-GFP Agrobacterium inoculation solution at a volume ratio of 2:1 to prepare the experimental treatment solution. Empty vector Agrobacterium inoculation solution was mixed with pSMV-GFP Agrobacterium inoculation solution in the same proportion to prepare the negative control solution.

[0032] Infiltration inoculation: Incubate the inoculum with the tobacco to be inoculated in the dark for at least 3 hours, then use a needleless syringe to inoculate the mixed bacterial solution (OD200). 600 Inoculate the tobacco leaf from the underside of the leaf with a solution of 0.5 μL (50 μL in volume), ensuring that the bacterial solution completely wets the inoculated area.

[0033] Culture: After inoculation, the plants were placed in a dark environment for 24 hours to facilitate Agrobacterium transformation, and then transferred to normal light conditions for continued culture.

[0034] ④ Results Observation and Conclusions Seven days after inoculation, the expression of GFP in the leaves was observed using a fluorescent protein monitoring lamp (GFP is expressed as a fusion of GFP and SMV polyprotein, therefore the GFP fluorescence represents the virus content). The observation results are as follows: Figure 5 and Figure 6 As shown in A, Glyma.13G184800 , Glyma.13G184900 , Glyma.13G187900 , Gs.13GNEW1 None of these four genes have an inhibitory effect on SMV. Figure 5), and transient overexpression GsRPP13L In tobacco leaves treated with the gene, the intensity of the GFP fluorescence signal was significantly weaker than that in the negative control group and other gene treatment groups. Figure 6 (A) in the text, prove GsRPP13L Gene expression effectively inhibited the replication and proliferation of SMV in Nicotiana benthamiana. Furthermore, qRT-PCR was used to detect... GsRPP13L The relative expression levels of SMV genomic RNA in leaves of the experimental group and the negative control group were detected as follows: Figure 6 As shown in B, compared to the negative control group, overexpression GsRPP13L The relative expression level of SMV genomic RNA was significantly reduced in the leaves of the experimental group, indicating transient overexpression. GsRPP13L The gene can significantly inhibit the proliferation of SMV in Nicotiana benthamiana.

[0035] Example 2: GsRPP13L Application of genes in improving soybean resistance to soybean mosaic virus 1. Construction of recombinant vectors Will GsRPP13L The CDS sequence of the gene was cloned downstream of the 35S promoter in the plant expression vector pCAMBIA3301 (nucleotide sequence shown in SEQ ID NO. 10) to construct a 35S-driven gene. GsRPP13L The gene overexpression vector pCAMBIA3301-GsRPP13L was constructed and validated by a biotechnology company. The product provided is the final product after successful sequencing. A schematic diagram of the vector's construction is shown below. Figure 7 As shown, the identification results of agarose gel electrophoresis are as follows: Figure 8 As shown, the primer pair used for identification consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 (5'-CTGACGTAAGGGATGACGC-3') and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8 (5'-GTTTAAAGAAAGATCAAAGCTC-3').

[0036] 2. Agrobacterium-mediated genetic transformation of soybean variety Dongnong 50 (1) Seed sterilization and pretreatment: Select plump, healthy soybean seeds of the Dongnong 50 variety with no cracks on the surface, sterilize them with chlorine in a fume hood for 13 hours; soak the sterilized seeds in sterile water in a clean bench for 4 hours (25℃, dark incubation); then cut the two cotyledons apart with a sterile blade and make 1-2 shallow cuts (depth ≤0.5 mm) near the hilum to create wounds that facilitate Agrobacterium infection.

[0037] (2) Preparation of Agrobacterium infection solution: The recombinant plasmid pCAMBIA3301-GsRPP13L obtained in step 1 was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium, named EHA105 / pCAMBIA3301-GsRPP13L. A single colony of recombinant Agrobacterium EHA105 / pCAMBIA3301-GsRPP13L was selected and inoculated into LB liquid medium containing 50 mg / L kanamycin. The culture was then incubated at 28°C with shaking at 200 rpm until the OD value of the bacterial culture reached a certain level. 600 The OD value reached 0.6-0.8. Afterwards, the cultured bacterial suspension was centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. Next, the bacterial cells were resuspended in infection liquid medium (LB medium containing 100 μM acetylsylcholine) to adjust the OD value of the bacterial suspension. 600 When the value reaches 0.3, the Agrobacterium infection solution is obtained.

[0038] (3) Infection and co-cultivation Soybean seeds pretreated in step (1) were immersed in the Agrobacterium infection solution obtained in step (2) and cultured at 28°C and 100 rpm for 30 min with shaking. The seeds were removed, excess bacterial solution was absorbed with sterile filter paper, and the seeds were placed on a co-culture medium (containing acetylsuccinone) and cultured in the dark at 22°C for 48 hours.

[0039] (4) Recovery and screening After co-culturing, the seeds were thoroughly rinsed three times with sterile water, dried, and then transferred to recovery medium. They were cultured at 25°C with a photoperiod of 16 hours of light and 8 hours of darkness for 7 days to inhibit the excessive growth of Agrobacterium. The recovered explants were then transferred to selection medium (containing appropriate antibiotics, such as glufosinate), and the medium was replaced with fresh medium every two weeks. The selection was continued for 4-6 weeks, and untransformed tissues were discarded.

[0040] (5) Regeneration and transplantation The surviving resistant tissues were transferred to the shoot induction medium and cultured under photoperiod until the adventitious shoots grew to 2-4 cm. Healthy shoot tips were cut off and transferred to the rooting medium to induce rooting (about 2-3 weeks). After rooting, the seedlings were hardened off for 3 days, the culture medium was washed off the roots, and the seedlings were transplanted into sterile nutrient soil. The seedlings were covered with a film to keep them moist for 3-5 days before normal management.

[0041] A total of 4 strains were obtained through the above genetic transformation process. GsRPP13L Gene overexpression lines were identified as OE1, OE2, OE3, and OE4. The effects of these four lines on... GsRPP13L Gene expression levels were validated, and the results were as follows: Figure 9 As shown, the four strains GsRPP13L Gene expression levels were 3-5 times higher than those of wild-type Dongnong 50.

[0042] 3. Overexpression GsRPP13L Verification of SMV resistance in genetically modified soybeans Plant virus infection in host plants is always "systemic infection," meaning that after a virus is inoculated onto a leaf of the plant (the inoculated leaf), the virus completes initial infection on that leaf, multiplies intracellularly, and then moves to adjacent cells via plasmodesmata. Finally, it moves to other tissues and organs of the plant, such as stems, roots, and other leaves, through the vascular bundle. Therefore, after a period of time (approximately 2-3 weeks) after inoculation, the virus will spontaneously migrate to the newly grown leaves (the "systemic leaves"). Thus, when evaluating plant resistance to viruses, the virus content in the inoculated leaf is usually measured one week after inoculation, and the virus content in the systemic leaves is measured three weeks after inoculation.

[0043] Wild-type susceptible soybean variety DN50 (WT) and four overexpressing genes were compared. GsRPP13L The DN50 transgenic lines (OE1, OE2, OE3, and OE4) were simultaneously sown in pots and cultured under the same conditions until the first round of trifoliate leaves unfolded. SMV-GFP was then inoculated onto the trifoliate leaves by friction. The specific inoculation method is as follows: Take leaves of frozen or live SMV-GFP source plants, add PBS buffer at pH 7.0 at a ratio of 1:10 (m / v) and grind them into a homogenate as the inoculation solution. Inoculate the soybean leaves by friction, with 100 μL per leaf.

[0044] Seven days after inoculation, inoculated leaves from WT, OE1, OE2, OE3, and OE4 plants were collected and designated as WT plant inoculated leaf, OE#1, OE#2, OE#3, and OE#4, respectively. GFP expression was observed using a fluorescent protein observation lamp. The results are shown below. Figure 10 A. Total RNA was extracted from inoculated leaf samples. Using the genomic RNA content in WT inoculated leaf samples as a baseline, the SMV genomic RNA content in the inoculated leaves was quantitatively analyzed using qRT-PCR. The results are shown in [Figure A]. Figure 10 B in the middle.

[0045] Twenty-one days after inoculation, systematic leaves were collected from WT, OE1, OE2, OE3, and OE4 plants, and designated as WT plant systematic leaves, OE#S1, OE#S2, OE#S3, and OE#S4, respectively. GFP expression was observed using a fluorescent protein monitoring lamp. The results are shown below. Figure 10 C. Total RNA was extracted from the leaf samples of the WT system. Using the genomic RNA content in the leaf samples of the WT system as a benchmark, the SMV genomic RNA content in the system leaves was analyzed by relative quantification using qRT-PCR. The results are shown in [Figure number missing]. Figure 10 D in the middle.

[0046] Figure 10 B and Figure 10The two graphs in Figure D represent the virus content in the inoculated leaves of WT, OE1, OE2, OE3, and OE4 plants 7 days after inoculation and the systemic leaves 21 days after inoculation, respectively. These are two independent experiments. The virus content data in OE#S1, OE#S2, OE#S3, and OE#S4 are analyzed based on the systemic leaves of WT plants, while the virus content data in OE#1, OE#2, OE#3, and OE#4 are analyzed based on the inoculated leaves of WT plants. Therefore, the relative expression level of SMV genomic RNA in both the systemic leaves and inoculated leaves of WT plants is 1.0. This value is a defined value, not a measured value. The fact that both are 1.0 does not mean that their virus content is consistent.

[0047] Regarding overexpression GsRPP13L Explanation of the difference in virus inhibition levels between leaves and inoculated leaves in transgenic soybean plant systems: The infection process of plant viruses in host plants can be divided into three main stages: The first stage, initial infection, occurs when the virus enters the leaf cells of the inoculated leaf directly through micro-wounds created by friction and undergoes early proliferation within the initially infected cells. The second stage is intercellular movement, which is the movement of progeny viruses proliferating within a single cell at the inoculation site to adjacent cells via plasmodesmata. The third stage is systemic movement, where the virus, which reaches the phloem cells via intercellular movement, rapidly moves from the inoculated leaf to the roots, stems, flowers, fruits, and other organs such as systemic leaves through the phloem's transport tissues.

[0048] In this embodiment, overexpression GsRPP13L The viral load in the inoculated leaves of genetically modified soybean plants was reduced by approximately 50%-70% compared to the wild type. Figure 10 (B in the text), this is due to GsRPP13L This is due to the inhibitory effect of the gene on the SMV virus in the first and second stages of viral infection. Overexpression... GsRPP13L The viral load in the systemic leaves of genetically modified soybean plants was reduced by approximately 80%-90% compared to the wild type. Figure 10 The D in the gene showed a stronger viral suppression effect. This is because the disease-resistant gene... GsRPP13L It is expressed not only in the leaves of transgenic plants but also in phloem cells, resulting in transgenic plants inhibiting the virus not only in the first and second stages but also in the third stage of viral infection (i.e., systemic viral movement), thus the systemic leaves show a stronger inhibitory effect.

[0049] The above results indicate that overexpression GsRPP13LThe levels of GFP fluorescence and SMV genomic RNA in the inoculated leaves and systemic leaves of transgenic soybean lines were significantly lower than those in wild-type Dongnong 50, indicating that... GsRPP13L The gene can inhibit the proliferation of SMV in soybeans.

Claims

1. A soybean for improving resistance to soybean mosaic virus. GsRPP13L Genes, characterized by, The soybean GsRPP13L The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The soybean according to claim 1 GsRPP13L Application of genes in enhancing resistance of tobacco or soybean to soybean mosaic virus.

3. The application according to claim 2, characterized in that, The application involves overexpressing soybean in tobacco. GsRPP13L Genes that enhance tobacco resistance to soybean mosaic virus, or overexpress soybean in soybeans. GsRPP13L Genes enhance soybean resistance to soybean mosaic virus.

4. A type of soybean containing the soybean of claim 1 GsRPP13L Gene recombination vectors.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector's vector skeleton is pCB301 or pCAMBIA3301.

6. The use of the recombinant vector according to any one of claims 4 or 5 in improving the resistance of tobacco or soybean to soybean mosaic virus.

7. A type of soybean comprising the soybean of claim 1 GsRPP13L Recombinant bacteria.

8. The recombinant bacteria according to claim 7, characterized in that, The recombinant bacteria originated from Agrobacterium.

9. The recombinant bacteria according to claim 8, characterized in that, The Agrobacterium is Agrobacterium EHA105 or Agrobacterium GV3101.

10. The use of the recombinant bacteria according to any one of claims 7-9 in improving the resistance of tobacco or soybean to soybean mosaic virus.

Images