Soybean gmst3d gene mutant and application thereof in regulating resistance of soybean to sclerotinia sclerotiorum
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-10
- Publication Date
- 2026-08-11
AI Technical Summary
本发明首次揭示了大豆GmST3d蛋白及其编码基因具有调控大豆对核盘菌抗性的功能，通过在植物中导入大豆GmST3d基因突变体可以有效提高大豆对核盘菌的抗性。本发明提供的GmST3d蛋白及其编码基因可以用于大豆抗性品种的选育，具有广阔的应用前景。
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Figure CN121674425B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and more particularly to soybeans. GmST3d Gene mutants and their application in regulating soybean resistance to Sclerotinia sclerotiorum. Background Technology
[0002] Soybeans are an important food and oilseed crop, but with changes in global climate and agricultural practices in recent years, soybean yields face significant challenges. Soybeans are threatened by various diseases during their growth, among which sclerotinia rot caused by Sclerotinia sclerotiorum is one of the major diseases threatening soybean yields.
[0003] Sclerotinia sclerotiorum is a pathogenic microorganism with an extremely wide host range. It can infect a variety of plants and can also transfect soybeans from various plants. In severe cases, it can cause the roots, stems, pods and seeds of soybeans to rot, and even cause the death of soybean plants, resulting in irreversible and serious damage to soybean yield.
[0004] Therefore, improving soybean resistance to Sclerotinia sclerotiorum is one of the important directions for increasing soybean yield. Discovering key genes that can regulate soybean resistance to Sclerotinia sclerotiorum has become a pressing technical challenge in this field. Summary of the Invention
[0005] To solve the above-mentioned technical problems, the present invention first provides soybeans. GmST3d The gene mutant has the nucleotide sequence shown in SEQ ID NO.3.
[0006] This invention has found that the mutant gene and its encoded protein can regulate soybean resistance to Sclerotinia sclerotiorum, and that the resistance to Sclerotinia sclerotiorum is negatively regulated.
[0007] Furthermore, the present invention provides a product containing the aforementioned soybean. GmST3d Biomaterials containing gene mutants.
[0008] In some embodiments, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.
[0009] Furthermore, this invention provides the application of soybean GmST3d protein or its encoding gene, or biological materials containing its encoding gene, in regulating soybean resistance to Sclerotinia sclerotiorum.
[0010] Preferably, the amino acid sequence of the soybean GmST3d protein is as shown in SEQ ID NO.1 or SEQ ID NO.10.
[0011] In specific implementation, proteins that have the same function as the amino acid sequence shown in SEQ ID NO.1 by substituting, deleting, or adding one or more amino acids are also within the scope of protection of this invention.
[0012] Preferably, the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.2 or SEQ ID NO.3.
[0013] SEQ ID NO.2 is GmST3d The CDS sequence of the gene, SEQ ID NO.3 is GmST3d Gene mutant, SEQ ID NO.10 is GmST3d The amino acid sequence of the protein encoded by the gene mutant.
[0014] In some embodiments, soybean resistance to Sclerotinia sclerotiorum is improved by reducing the activity or expression level of soybean GmST3d protein or its encoding gene; the amino acid sequence of said soybean GmST3d protein is SEQ ID NO.1.
[0015] Furthermore, this invention provides the application of soybean GmST3d protein or its encoding gene, or biological materials containing its encoding gene, in the preparation of transgenic soybeans.
[0016] Furthermore, this invention provides the application of soybean GmST3d protein or its encoding gene, or biological materials containing its encoding gene, in soybean breeding.
[0017] In some implementations, the breeding methods include, but are not limited to, transgenic, hybrid, backcross, self-pollination, or asexual reproduction.
[0018] Preferably, the purpose of the soybean breeding is to cultivate new soybean varieties resistant to Sclerotinia sclerotiorum.
[0019] Furthermore, the present invention provides a method for improving soybean resistance to Sclerotinia sclerotiorum, comprising: reducing the activity or expression level of soybean GmST3d protein or its encoding gene to improve soybean resistance to Sclerotinia sclerotiorum; wherein the amino acid sequence of soybean GmST3d protein is SEQ ID NO.1.
[0020] Preferably, the method includes: modifying the GmST3d protein or its encoding gene to render the GmST3d protein or its encoding gene nonfunctional, thereby improving the resistance of soybean to Sclerotinia sclerotiorum; the amino acid sequence of the soybean GmST3d protein is SEQ ID NO.1.
[0021] Preferably, the resistance of soybean to Sclerotinia sclerotiorum is improved by using genetic engineering to reduce the activity or expression level of soybean GmST3d protein or its encoding gene; the amino acid sequence of soybean GmST3d protein is SEQ ID NO.1.
[0022] Preferably, soybeans are edited using the CRISPR-Cas9 system. GmST3d The gene reduces the activity or expression level of soybean GmST3d protein or its encoding gene to improve soybean resistance to Sclerotinia sclerotiorum; the amino acid sequence of the soybean GmST3d protein is SEQ ID NO.1.
[0023] Preferably, the method includes: using soybeans GmST3d Using the gene (SEQ ID NO.2) as the target, a CRISPR-Cas9-based knockout target sequence was designed. An oligonucleotide sequence encoding the knockout target sequence was first linked into the pBlu gRNA intermediate vector to construct gRNA. The constructed gRNA was then digested with enzymes, and the digested gRNA was ligated into a CRISPR-Cas9 vector, transformed into soybean, and thus obtained... GmST3d Genetically modified soybeans with missing gene function.
[0024] Preferably, the knockout target sequence is shown in SEQ ID NO.4.
[0025] Preferably, the sequence of the gRNA is shown in SEQ ID NO.5.
[0026] Preferably, the method includes: placing the soybeans... GmST3d Gene mutants are introduced into soybeans via vectors or integrated into soybean chromosomes through genetic engineering.
[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention reveals for the first time that the soybean GmST3d protein and its encoding gene have the function of regulating soybean resistance to Sclerotinia sclerotiorum, and this can be achieved by introducing soybean into the plant. GmST3d Gene mutants can effectively enhance soybean resistance to Sclerotinia sclerotiorum. The GmST3d protein and its encoding gene provided by this invention can be used for the breeding of resistant soybean varieties and have broad application prospects. Attached Figure Description
[0028] Figure 1 For some soybeans GmST3d The genome and transcription sequence of a gene; where bold indicates exon sequences, italics indicate intron sequences, ATG indicates start codon, and TGA indicates stop codon.
[0029] Figure 2 To buildGmST3d Diagram of the intermediate vector used in CRISPR-Cas9 gene knockout plants.
[0030] Figure 3 To build GmST3d A diagram of the CRISPR-Cas9 vector used in CRISPR-Cas9 knockout plants.
[0031] Figure 4 To build GmST3d Gene knockout GmST3d -1 mutant plants GmST3d Diagram of gene mutation patterns.
[0032] Figure 5 for GmST3d The graph shows the results of resistance detection to Sclerotinia sclerotiorum in mutant plants and wild-type Williams 82 soybean, where A represents... GmST3d Mutant plants ( GmST3d-1 Phenotypic images of resistance to Sclerotinia sclerotiorum and wild-type Williams 82 soybean (WT), B is a statistical graph of lesion area. This indicates that there is a statistically significant difference. Detailed Implementation
[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0034] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. Furthermore, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as gene sequencing, plasmid sequencing, and molecular weight determination.
[0035] Example 1 This embodiment focuses on soybeans. GmST3d Gene structure analysis includes the following procedures: 1. GmST3d gene segregation Total RNA was extracted from the soybean variety Williams 82. Using the total RNA as a template and oligonucleotide sequence (T)17 as a primer, the first strand of cDNA was synthesized. Using this first strand of cDNA as a template, PCR amplification was performed using both the forward primer (5'-ATGGCAGAGACCATTCGTTTG-3', SEQ ID NO. 6) and the reverse primer (5'-ACATGAACGGACACTCGTGA-3', SEQ ID NO. 7). A 765bp cDNA was obtained. GmST3d The gene cDNA fragment (SEQ ID NO. 2) was ligated to the pEasy-Blunt vector (Kangrun Biotechnology Co., Ltd.), and named Blunt- GmST3d .
[0036] The embodiment obtained GmST3d The gene, whose CDS sequence is shown in SEQ ID NO.2; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1.
[0037] 2. GmST3d Gene structure analysis DNA was extracted from young leaves of the soybean variety Williams 82, and amplified using this genomic DNA as a template. GmST3d Genome fragments, part GmST3d Genome sequences such as Figure 1 As shown, the total length is 1877bp, containing 6 exons and 5 introns.
[0038] Example 2 This embodiment is constructed using CRISPR-Cas9 technology. GmST3d mutant ( GmST3d-1 The specific process is as follows: 1. Design GmST3d Gene knockout target sequence Search in the Arabidopsis database (www.arabidopsis.org) GmST3d Genes were analyzed to obtain their amino acid sequences. The BLAST tool (https: / / phytozome-next.jgi.doe.gov / blast-search) was used in... Glycine maxSearch for corresponding homologous genes in the soybean genome. Input the corresponding genes using the CRISPR design tool (http: / / cfans-pmorrell.oit.umn.edu / CRISPR / ). GmST3d The DNA sequence was determined. Target oligonucleotide sequences containing sticky ends were designed and synthesized. The oligonucleotide sequences are as follows: F:5'-GATTGGTGTTTCAGTAACAATGTA-3' (SEQ ID NO.8) F:5'-AAAACTACATTGTTACTGAAACACC-3' (SEQ ID NO.9) The target sequence is: 5'-GGTGTTTCAGTAACAATGTA-3' (SEQ ID NO.4) 2. gRNA cloning The oligonucleotide sequence of the target site was diluted and annealed (50℃, 6 hours), and the pBlu gRNA vector was digested with BbsI (https: / / stuparlab.cfans.umn.edu / protocols / crisprcas9-glycine-max). Figure 2 The enzyme digestion products were separated by gel electrophoresis, and a 3500 bp vector fragment was extracted. The annealed oligonucleotide sequence was ligated to the digested pBlu gRNA vector (using T4 ligase, overnight at 16°C). The ligation product was transformed into *E. coli* (Top10), and positive clones were screened. The plasmid was extracted and sequenced (using T3 primer 5'-aattaaccctcactaaaggg-3', SEQ ID NO. 11) to confirm the correct gRNA sequence.
[0039] 3. gRNA insertion into the Cas9 vector The validated pBlu gRNA vector and the target Cas9 vector were digested with EcoRI. The digestion products were separated by gel electrophoresis, and a 554 bp gRNA fragment (SEQ ID NO. 5) was extracted. The gRNA fragment was then further mixed with the digested Cas9 vector (…). Figure 3 The ligation was performed using T4 ligase (https: / / stuparlab.cfans.umn.edu / protocols / crisprcas9-glycine-max), overnight at 16°C. The ligation product was transformed into *E. coli* (DH5α), and positive clones were screened. Colony PCR was used to screen positive clones to confirm correct gRNA fragment insertion. Plasmids were extracted and sequenced to ensure correct CRISPR-Cas9 vector construction.
[0040] 4. Transform soybeans and screen for positive plants. The constructed CRISPR-Cas9 vector was introduced into Williams 82 soybean callus via Agrobacterium-mediated transformation. Positive transformants were selected using the Bar cassette selection marker. Genomic DNA was extracted from the transformants, and the knockout of the target gene was verified by PCR and sequencing to confirm whether they were homozygous knockout plants. GmST3d mutant plants GmST3d-1 The knockout results are as follows Figure 4 As shown. GmST3d In mutant plants, GmST3d The nucleotide sequence of the gene mutant is shown in SEQ ID NO.3.
[0041] Example 3 GmST3d mutant plants ( GmST3d-1 The resistance of Williams 82 plants to Sclerotinia sclerotiorum was tested, with Williams 82 plants used as a control (WT). The specific method is as follows: Williams 82 and GmST3d-1 Young true leaves at 10 days old were inoculated with Sclerotinia sclerotiorum activated for three days. The area of lesions on the leaves was counted three days after inoculation. At least 5 plants were prepared for each planting, and 3 true leaves from different plants were inoculated each time. The inoculation was repeated three times, and the average of the three replicates was taken as the area of lesions for that soybean variety.
[0042] Williams 82 and GmST3d-1 Five individual plants were planted. After 10 days of growth, three true leaves from each variety were taken to measure the area of lesions after inoculation with *Sclerotinia sclerotiorum*. The average value of the measurements from all leaves for each variety was taken as the lesion area for that soybean variety. The results are as follows: Figure 5 As shown.
[0043] The experimental results above show that, GmST3d-1 The lesion area in the control group was significantly smaller than that in the control group (Williams 82), indicating that... GmST3d-1 Enhanced resistance to Sclerotinia sclerotiorum; the GmST3d protein negatively regulates soybean resistance to Sclerotinia sclerotiorum. Therefore, soybean... GmST3d Gene mutants can effectively improve soybean resistance to Sclerotinia sclerotiorum, and the GmST3d protein and its encoding gene can serve as new targets for soybean disease resistance breeding.
[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. The application of soybean GmST3d protein or its encoding gene, or biological material containing its encoding gene, in regulating soybean resistance to Sclerotinia sclerotiorum; the application improves soybean resistance to Sclerotinia sclerotiorum by reducing the activity or expression level of soybean GmST3d protein or its encoding gene; the amino acid sequence of soybean GmST3d protein is SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.
2.
3. The application of a method for reducing the activity or expression of soybean GmST3d protein or its encoding gene in improving soybean resistance to Sclerotinia sclerotiorum, wherein the amino acid sequence of soybean GmST3d protein is SEQ ID NO.
1.
4. The application of soybean GmST3d protein or its encoding gene, or biological material containing its encoding gene, in the preparation of transgenic soybeans; the application improves the resistance of soybeans to Sclerotinia sclerotiorum by reducing the activity or expression level of soybean GmST3d protein or its encoding gene; the amino acid sequence of soybean GmST3d protein is SEQ ID NO.
1.
5. The application of soybean GmST3d protein or its encoding gene, or biological materials containing its encoding gene, in soybean breeding; the application improves soybean resistance to Sclerotinia sclerotiorum by reducing the activity or expression level of soybean GmST3d protein or its encoding gene; the amino acid sequence of soybean GmST3d protein is SEQ ID NO.
1.
6. A method for improving soybean resistance to Sclerotinia sclerotiorum, characterized in that, include: The resistance of soybean to Sclerotinia sclerotiorum was improved by reducing the activity or expression level of soybean GmST3d protein or its encoding gene; the amino acid sequence of soybean GmST3d protein is SEQ ID NO.1.