Application of wild soybean GsIFS2 coding gene in resisting soybean mosaic virus disease

Abstract

The invention discloses an application of a soybean GsIFS2 coding gene in soybean mosaic virus disease resistance. The invention discloses application of the soybean GsIFS2 gene as shown in SEQ ID NO.1 in genetic engineering modification of soybean mosaic virus resistance. The over-expression of the gene can significantly improve the resistance of soybeans to soybean mosaic virus diseases. The soybean coding gene GsIFS2 disclosed by the invention can be transformed into soybeans through genetic engineering, and the resistance of the soybeans to soybean mosaic virus diseases is finally regulated and controlled.

Description

Technical Field

[0001] The invention relates to the application of wild soybean GsIFS2 coding gene in resistance to soybean mosaic virus disease, belongs to the field of genetic engineering, and specifically to the application of wild soybean GsIFS2 in changing resistance to soybean mosaic virus. Background Art

[0002] Soybean is an important grain and oil crop, providing high-quality vegetable oils and proteins for humans and animals. Soybean is harmed by a variety of diseases and pests throughout its growth period, which seriously affects the yield and quality of soybeans. Among these diseases, soybean mosaic virus disease is caused by soybean mosaic virus (SMV), which is a global disease that occurs in almost all soybean producing areas. Soybeans infected with SMV show symptoms such as mosaic leaves, yellow spotted mosaic leaves, curled leaves, deformed and wrinkled leaves, brown spotted seeds, pod deformities, reduced number of pods per plant, reduced plant height, reduced branches, and delayed growth. Soybean resistance to SMV is divided into resistance to infection and resistance to expansion. Resistance to infection means that the pathogen cannot infect the host, which belongs to quality resistance or vertical resistance, an extreme resistance. If a single resistant material is planted for a long time, facing high selection pressure, the physiological subspecies of the pathogen will produce new mutations, and the original resistant material will become susceptible material, thus lacking resistance materials to the newly mutated subspecies or strains, which will cause field resistance loss. Resistance to expansion means that the pathogen can successfully infect the host and establish a parasitic relationship, but the host can produce a defensive response to the pathogen, secrete antitoxins or inhibit the replication and spread of the pathogen, reduce the reproduction of the pathogen, and reduce the harm. It belongs to quantitative resistance or horizontal resistance. Varieties with quantitative resistance will not cause selection pressure on pathogens, are not easy to lose resistance, and can still achieve good yields in the case of a major outbreak of the disease. At present, breeding soybeans resistant to SMV is the most economical and effective measure to control SMV. Discovering disease-resistant genes related to the two resistance mechanisms is crucial to improving soybean resistance to SMV and breeding disease-resistant varieties.

[0003] In the early stage, by analyzing the SMV-induced soybean transcriptome, it was found that GsIFS2 strongly responded to the induction of the soybean mosaic virus SC7 strain. GsIFS2 is a key enzyme in the isoflavone biosynthesis pathway, converting flavonoid substrates into isoflavone products. Wild soybean IFS has two homologous genes, GsIFS1 and GsIFS2, and the expression level of GsIFS2 is positively correlated with isoflavone accumulation. When RNA interference IFS is used in soybeans, the biosynthesis of soybean isoflavones is blocked and isoflavones no longer accumulate. Whether GsIFS2 is resistant to soybean mosaic virus disease in soybeans has not been reported. Summary of the invention

[0004] The purpose of the present invention is to disclose the application of wild soybean isoflavone synthase gene GsIFS2 in genetic engineering for SMV resistance. The gene can be introduced into soybean as a target gene to regulate the resistance of soybean to SMV.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The soybean isoflavone synthase gene GsIFS2 has a nucleotide sequence as shown in SEQ ID NO.1 and an amino acid sequence as shown in SEQ ID NO.2.

[0007] The application is preferably to overexpress GsIFS2 in soybean to reduce the accumulation of SMV in soybean.

[0008] When using GsIFS2 to construct a plant overexpression vector, any enhanced promoter or inducible promoter can be added before its transcription start nucleotide. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding a selective marker gene (GUS gene, luciferase gene, etc.) to the plant. From the perspective of the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be screened by adversity.

[0009] After the wild soybean isoflavone synthase encoding gene GsIFS2 of the present invention is transformed into soybeans through genetic engineering, the gene is overexpressed to reduce the SMV content in the soybeans.

[0010] The plant overexpression vector carrying the GsIFS2 of the present invention can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, DNA direct transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants. The transformed plant host can be monocotyledonous plants such as sorghum, rice, wheat, and corn, or dicotyledonous plants such as peanut, soybean, rapeseed, tomato, poplar, lawn grass, and alfalfa.

[0011] The invention discloses an application of a vector for overexpressing the soybean GsIFS2 gene in improving the resistance of soybean to soybean mosaic virus disease.

[0012] Introducing a vector that overexpresses the soybean GsIFS2 gene into soybean can reduce the amount of SMV accumulation in soybean.

[0013] Beneficial effects:

[0014] Wild soybean GsIFS2 is a gene encoding isoflavone synthase. We found that the GsIFS2 gene in soybean responds to SMV induction, and its expression level is significantly increased and the SMV content is reduced in overexpressed transgenic soybeans. Therefore, GsIFS2 can be used to breed soybean varieties resistant to SMV. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 .GsIFS2 gene was induced in response to soybean mosaic virus SC7. HAAS_075: susceptible material, NJAU_W052: resistant material. *, **, *** indicate significant differences at 0.05, 0.01 and 0.001 levels, respectively.

[0016] Figure 2 .EPSPS test strip test results of GsIFS2 overexpressing transgenic soybeans. A. Test results of GsIFS2 overexpressing transgenic soybean line IFS-29-4, B. Test results of GsIFS2 overexpressing transgenic soybean line IFS-43-3. The first one on the left of the two groups of pictures is the control Jack, and the rest are GsIFS2 overexpressing transgenic soybeans

[0017] Figure 3 .Relative expression of GsIFS2 gene in overexpressed transgenic soybean. Jack is wild type, IFS-29-4 and IFS-43-3 represent two GsIFS2 overexpressed transgenic soybean lines, three biological means ± standard error (SE), ** and **** indicate significance at 0.01 and 0.0001 levels, respectively.

[0018] Figure 4 .Leaves of GsIFS2-overexpressing transgenic soybeans inoculated with SMV-SC7 strain 21 days after inoculation. PBS is the control, 0.01 mol / L phosphate buffer was applied, SC7 is inoculated with SMV SC7 strain, and the legend is 1 cm.

[0019] Figure 5 .Determination of the expression level of SMV virus CP protein gene in GsIFS2 overexpressing transgenic soybean. Jack is wild type, IFS-

[0020] 29-4 and IFS-43-3 represent two GsIFS2 overexpressing transgenic soybean lines. qRT-PCR was used to detect the expression of SMV virus CP gene. Three biological means ± standard error (SE), * indicates significant at the 0.05 level. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0022] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0023] Example 1 Induction of the GsIFS2 gene in response to soybean mosaic virus SC7 strain

[0024] The susceptible material HAAS_075 and the disease-resistant material NJAU_W052 were planted, and the soybean mosaic virus SC7 strain was inoculated at the true leaf stage. The soybean leaf tissue was taken 0, 2, 8, and 24 hours after inoculation, ground with a mortar, added to a 1.5 mL EP tube containing lysis solution, and after sufficient shaking, transferred to a 1.5 mL EP tube to extract total RNA (Total RNA Kit, Tiangen, Beijing, China). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and the RNA content was determined by spectrophotometer. GsTubulin constitutively expressed in wild soybean was used as an internal reference, and the primer sequences were F: 5'-GGAGTTCACAGAGGCAGAG-3' and R: 5'-CACTTACGCATCACATAGCA-3'. The obtained total RNA was used as a template, and the reverse transcription kit (TaKaRa PrimerScript TM RT reagent kit, Japan) was used for reverse transcription to obtain the first strand of cDNA, and then real-time RT-PCR was performed to detect the expression changes of GsIFS2 gene at different time points after the two materials were infected with the virus. The primer sequences were F: 5'-GGGTCTTGTTGTCGACTTTTT-3' and R: 5'-ATGTCTCCTTCACGATTGCTC-3'. The results showed that GsIFS2 gene strongly responded to the induction of soybean mosaic virus SC7 strain in both materials at each time point ( Figure 1 ).

[0025] Example 2 Genetic Engineering Application of Gene GsIFS2

[0026] 1) Cloning of wild soybean GsIFS2

[0027] Using the mRNA sequence of the Glysoja.11G030998 (GsIFS2) gene of the soybean reference genome W05 in the SoyBase database as a template, specific primers were designed, and the primer sequences for amplifying the gene were F: 5'-ATGTTGCTTGAACTTGCACTTG-3' and R: 5'-TAAGAAAGGAGTTTAGATGCAA-3'. PCR amplification was performed using the leaf cDNA of the wild soybean variety ZYD4368 as a template, and the total RNA extraction from the leaves and the reverse transcription of the first chain of cDNA were the same as in Example 1. The PCR program was as follows: 95°C pre-denaturation for 30 seconds, 95°C denaturation for 10 seconds, 60°C annealing for 20 seconds, 72°C extension for 30 seconds, a total of 35 cycles, and finally 72°C incubation for 5 minutes. The PCR product was cloned into the PUC19-T vector, and the CDS sequence of the soybean GsIFS2 gene with a complete coding region of 1566 bp was obtained after sequencing, wherein the coding region sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0028] 2) Construction of plant expression vector

[0029] The GsIFS2 gene sequence was extended from the PUC19-T recombinant vector using PCR reaction, and the primers were: F: 5'-ATGTTGCTTGAACTTGCACTTG-3' and R: 5'-TAAGAAAGGAGTTTAGATGCAA-3'. The ligase-independent single-fragment one-step cloning kit ( Entry One Step Cloning Kit) was used to connect GsIFS2 to the plant expression vector D60005 (Da Bei Nong Group) to obtain a plant overexpression recombinant vector containing the GsIFS2 gene, which contains the marker gene EPSPS gene. The plant transformation vector D60005 contains a 2×35S strong promoter, which can strongly promote the expression of the target gene GsIFS2 in the recipient.

[0030] 3) Phenotypic identification

[0031] Two GsIFS2 overexpressing transgenic soybean lines were obtained, named IFS-29-4 and IFS-43-3. GsIFS2 gene overexpressing transgenic soybeans were planted in a constant temperature and light incubator. Leaves were taken after 7 days for EPSPS test strip detection and GsIFS2 gene expression detection. The EPSPS test strip detection results showed that the GsIFS2 gene had been successfully inserted into the transgenic soybeans ( Figure 2 ). The extraction, reverse transcription and fluorescence quantitative PCR reaction of total RNA are the same as those in Example 1) to detect the changes in the expression of the GsIFS2 gene in transgenic soybeans. Figure 3It can be seen that the expression level of GsIFS2 in the overexpressing transgenic soybean is significantly higher than that in the wild type (JACK).

[0032] After the first pair of true leaves unfolded, wild-type Jack and overexpression strains (IFS-29-4 and IFS-43-3) were inoculated with SMV-SC7 virus. The disease was observed 21 days after inoculation, and the expression of viral CP gene was detected in leaves. The leaves of wild-type Jack plants showed obvious wrinkling, while the leaves of overexpression strains showed mild mosaic symptoms ( Figure 4 ). Analysis of the expression of the viral CP gene revealed that the expression of the SMV-CP gene in the two overexpression strains was significantly lower than that in the wild type ( Figure 5 ). The above results showed that overexpression of GsIFS2 gene reduced the accumulation of soybean mosaic virus and improved the resistance of soybean to SMV-SC7. The primer sequences of CP gene were F: cagatgggcgtggttatga and R: acaatgggtttcagcggata.

Claims

1. Application of soybean GsIFS2 gene in genetic engineering to modify soybean resistance to soybean mosaic virus disease, wherein the coding region sequence of the soybean GsIFS2 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: Overexpression of soybean GsIFS2 gene can improve soybean resistance to soybean mosaic virus disease.

3. The use according to claim 1, characterized in that: Overexpression of GsIFS2 in soybean reduced SMV accumulation in soybean.

4. Use of a vector overexpressing the soybean GsIFS2 gene described in claim 1 in improving the resistance of soybean to soybean mosaic virus disease.

5. The use according to claim 4, characterized in that: Introducing a vector overexpressing the soybean GsIFS2 gene of claim 1 into soybean can reduce the amount of SMV accumulation in soybean.

Images