Construction method of soybean GmLNK2 gene four mutants and application of soybean GmLNK2 gene four mutants in soybean disease-resistant breeding

Abstract

The invention belongs to the technical field of plant genetic engineering and disease-resistant breeding, and particularly relates to a construction method of soybean GmLNK2 gene four mutants and application of the soybean GmLNK2 gene four mutants in soybean disease-resistant breeding, by means of a CRISPR / Cas9 technology, wild soybean WS82 is used as a material, three target spots are designed for GmLNK2a, GmLNK2b, GmLNK2c and GmLNK2d genes of a GmLNK2 gene family, and simultaneous knockout is performed to obtain a mutant plant. Compared with a wild type WS82, the plant height of a mutant plant is obviously reduced, the length of a main root is obviously shortened, and the leaf area of three-out compound leaves is obviously reduced, which indicates that the mutant plant is more sensitive to pathogenic bacteria in a root rot disease nursery, the susceptibility degree is more serious, and growth and development are obviously inhibited, the key effect of the GmLNK2 gene family in soybean root rot resistance is clarified, and the application of the GmLNK2 gene family in soybean root rot resistance is developed. Important gene resources and technical support are provided for soybean disease-resistant molecular mechanism research and disease-resistant variety cultivation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and crop disease resistance breeding, and specifically relates to the construction method of soybean mutants and their application in soybean disease resistance breeding. Background Technology

[0002] Soybeans are an important food crop, oilseed crop, and cash crop in my country, occupying a vital position in the national economy. However, soybeans are susceptible to various diseases and pests during their growth, among which root rot is a common soil-borne disease in soybean-producing areas, caused by various pathogens (such as Fusarium and Phytophthora). Root rot damages the absorption function of soybean roots, leading to stunted plant growth and development, manifested as dwarfing, yellowing leaves, and reduced yield. In severe cases, it can even cause plant death, resulting in significant economic losses to soybean production.

[0003] Breeding and promoting disease-resistant varieties is the most economical, effective, and environmentally friendly way to control soybean root rot. With the development of molecular biology techniques, the precise regulation of the expression or function of soybean disease-resistant genes through gene editing has become an important means of breeding new disease-resistant soybean varieties. CRISPR / Cas9 technology, due to its advantages of ease of operation, high editing efficiency, and strong targeting, has been widely used in plant gene function research and molecular breeding. For example, patent CN202510971414.7 discloses a gene that negatively regulates resistance to Phytophthora in soybean. GmCDK8 Knockout GmCDK8 The study significantly enhanced soybean plants' resistance to Phytophthora root rot. Genetic engineering improvements using this gene to combat Phytophthora root rot in plants not only provide important genetic resources for plant molecular breeding but also offer an effective means to achieve high yields. Therefore, identifying key soybean disease resistance genes and elucidating the molecular mechanisms of disease resistance are of significant theoretical and practical importance for breeding disease-resistant soybean varieties. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention proposes a soybean GmLNK2 Methods for constructing four mutant genes and their application in soybean disease resistance breeding.

[0005] The technical solution of this invention is implemented as follows: On the one hand, the present invention provides a method for knocking out soybeans. GmLNK2 Application of gene function in breeding disease-resistant soybean plants.

[0006] Preferably, the above GmLNK2 Genes include GmLNK2a Gene, GmLNK2b Gene, GmLNK2c Genes and GmLNK2d Gene.

[0007] Preferably, the aboveGmLNK2a The nucleotide sequence of the gene is shown in SEQ ID NO.1. GmLNK2b The nucleotide sequence of the gene is shown in SEQ ID NO.2. GmLNK2c The nucleotide sequence of the gene is shown in SEQ ID NO.3. GmLNK2d The nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0008] Preferably, the above knockout is achieved through RNA interference technology or CRISPR / Cas9 gene editing technology; the disease resistance is resistance to soybean root rot.

[0009] Secondly, the present invention also provides a soybean GmLNK2 Gene tetra mutant gmlnk2-4m The construction method and steps are as follows: (1) Can be knocked out simultaneously GmLNK2a , GmLNK2b , GmLNK2c and GmLNK2d Three specific targets of the gene were constructed into the CRISPR / Cas9 gene editing vector; (2) The CRISPR / Cas9 gene editing vector was transferred into soybean plants using Agrobacterium-mediated transformation. After screening and sequencing verification, homozygous tetra mutant plants were obtained. gmlnk2-4m .

[0010] Preferably, the above GmLNK2a The nucleotide sequence of the gene is shown in SEQ ID NO.1. GmLNK2b The nucleotide sequence of the gene is shown in SEQ ID NO.2. GmLNK2c The nucleotide sequence of the gene is shown in SEQ ID NO.3. GmLNK2d The nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0011] Preferably, the three specific targets are target 1, target 2 and target 3, with the sequence of target 1 as shown in SEQ ID NO.5, the sequence of target 2 as shown in SEQ ID NO.6 and the sequence of target 3 as shown in SEQ ID NO.7.

[0012] Preferably, the construction steps of the above-mentioned CRISPR / Cas9 gene editing vector are as follows: the designed target sequence is ligated downstream of the vector U6 promoter to construct a recombinant editing vector. The specific steps are: the vector is double-digested with BsaI restriction enzyme and the vector backbone is recovered; the target sequence is annealed to form a double-stranded DNA fragment, which is ligated with the vector backbone using T4 DNA ligase, transformed into E. coli DH5α competent cells, and single colonies are picked for PCR amplification and sequencing verification to obtain a positive recombinant editing vector.

[0013] Preferably, the above-mentioned Agrobacterium-mediated method involves introducing the recombinant editing vector into wild-type soybean WS82 using Agrobacterium-mediated cotyledonary node transformation. The specific steps are as follows: Wild-type WS82 soybean seeds are sterilized and germinated under aseptic conditions; 5-7 day old cotyledonary nodes are used as explants; Agrobacterium-containing bacterial solution carrying the recombinant editing vector is co-cultured with the explants for 3 days; the co-cultured explants are transferred to an induction medium containing selection markers for callus induction and shoot differentiation; when the differentiated shoots reach 3-5 cm in length, they are transferred to a rooting medium to induce rooting; after rooting, the seedlings are hardened off and transplanted into a greenhouse for cultivation to obtain transgenic soybean plants.

[0014] Genomic DNA was extracted from transgenic soybean plants, and PCR amplification was performed using specific primers flanking the target sites. The amplified products were then sequenced for analysis. Plants showing base deletions, insertions, or substitutions were further subjected to homozygous self-pollination. After two consecutive generations of self-pollination and sequencing verification, the resulting transgenic soybean plants were successfully isolated. GmLNK2a , GmLNK2b , GmLNK2c , GmLNK2d A plant with homozygous mutations in all four genes is called a plant. gmlnk2-4m Quad mutant Thirdly, the present invention also provides the application of the mutant constructed by the above-mentioned construction method in soybean disease resistance breeding, characterized in that: the disease resistance is resistance to soybean root rot.

[0015] The present invention has the following beneficial effects: This invention provides a soybean GmLNK2 Gene family four mutants gmlnk2-4m The construction method utilizes CRISPR / Cas9 gene editing technology to target... GmLNK2 Gene family GmLNK2a , GmLNK2b , GmLNK2c and GmLNK2d Three target sites were simultaneously knocked out using gene design, and mutant plants were obtained using wild-type soybean WS82 as material. gmlnk2-4m Wild-type WS82 and gmlnk2-4m The four mutants were planted in soybean root rot disease nurseries, and their physiological phenotypes were statistically analyzed at the initial flowering stage of soybean. The results showed that... gmlnk2-4m The tetramutant was 49% shorter in height, 51.4% shorter in taproot length, and 41.8% smaller in leaf area compared to the wild type. These phenotypic indicators demonstrate that... gmlnk2-4m The four mutants are more susceptible to pathogens in root rot nurseries, exhibiting more severe infection and consequently significantly inhibiting growth and development. This also clarifies... GmLNK2The key role of gene families in soybean resistance to root rot can serve as candidate genes for soybean disease resistance breeding. In addition, the method of simultaneously knocking out multiple genes using CRISPR / Cas9 technology adopted in this invention also provides a technical reference for the functional study of multi-gene families in other crops. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Wild type soybean GmLNK2 Nucleotide sequence alignment of the gene and the mutant gene; the top of the figure shows the wild type. GmLNK2a , GmLNK2b , GmLNK2c and GmLNK2d The sequence is shown below, and the mutant sequence is shown below.

[0018] Figure 2 For WS82 and in the disease nursery gmlnk2-4m Plant height statistics; Figure A shows the physiological phenotypic analysis, and Figure B shows the plant height measurement results.

[0019] Figure 3 For WS82 and in the disease nursery gmlnk2-4m Statistical analysis of taproot length; Figure A shows the physiological phenotypic analysis, and Figure B shows the taproot length measurement results.

[0020] Figure 4 For WS82 and in the disease nursery gmlnk2-4m The first trifoliate compound leaf leaf area statistics; Figure A shows the physiological phenotypic analysis, and Figure B shows the leaf area measurement results of the trifoliate compound leaf. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0023] The LNK (LOV KELCH PROTEIN) gene family is a plant-specific gene family, and existing research has shown that it plays an important role in plant growth and development, light signal transduction, and responses to abiotic stress. Soybean GmLNK2 The gene family contains multiple members, and the functions of these members in soybean disease resistance, especially resistance to root rot, are currently unclear. Therefore, constructing gene-edited organisms using gene editing technology is crucial. GmLNK2 Investigating the role of mutant gene families in soybean resistance to root rot is of great theoretical and practical significance for identifying key soybean disease resistance genes, elucidating the molecular mechanisms of disease resistance, and breeding disease-resistant varieties.

[0024] The invention constructs gmlnk2-4m The four mutants were identified. GmLNK2 The correlation between gene families and soybean resistance to root rot provides important experimental materials for a deeper understanding of the molecular mechanisms underlying soybean resistance to root rot. Meanwhile, GmLNK2 Gene families can serve as candidate genes for soybean disease resistance breeding. Precise regulation of these gene families (such as overexpression) holds promise for developing new soybean varieties with stronger resistance to root rot, which has significant application potential for ensuring soybean yield and quality. Furthermore, the CRISPR / Cas9 technology used in this invention to simultaneously knock out multiple genes provides a technical reference for functional studies of multi-gene families in other crops.

[0025] Example 1: Soybeans GmLNK2 Construction of gene family four mutants Using wild-type soybean WS82 as material, targeting soybean GmLNK2 Four members of the gene family GmLNK2a, GmLNK2b , GmLNK2c and GmLNK2d Three specific target sites were designed (target sequences were designed based on conserved gene regions to ensure simultaneous knockout of four genes), and a CRISPR / Cas9 gene editing vector was constructed. The editing vector was introduced into wild-type WS82 soybean using Agrobacterium-mediated soybean genetic transformation. After screening and sequencing verification, the desired results were obtained. GmLNK2a, GmLNK2b , GmLNK2c and GmLNK2d A homozygous tetramutant in which all four genes are mutated simultaneously is named gmlnk2-4m . soybean wild type GmLNK2 Gene family genes GmLNK2a , GmLNK2b , GmLNK2c, GmLNK2d Comparison with the nucleotide sequence of the mutant gene, such as Figure 1 As shown.

[0026] The specific steps are as follows: (1) Target design and editing vector construction According to soybeans GmLNK2a , GmLNK2b , GmLNK2c , GmLNK2d The CDS sequences of the genes were used to design three specific targets using the CRISPR-P 2.0 online tool. The target sequences are Target1: 5'-TAACATAATATGGGGTGA-3' (SEQ ID NO.5), Target2: 5'-AAAACTGATCAGGGTTCCCT-3' (SEQ ID NO.6), and Target3: 5'-TCATATTGTGCCTTATCCGG-3' (SEQ ID NO.7), ensuring that the three targets can simultaneously recognize and bind to the conserved regions of the four genes.

[0027] The designed target sequence was ligated downstream of the U6 promoter to construct a recombinant editing vector. The specific steps were as follows: the vector was double-digested with BsaI restriction enzyme, and the vector backbone was recovered; the target sequence was annealed to form a double-stranded DNA fragment, which was then ligated to the vector backbone using T4 DNA ligase. The fragment was transformed into *E. coli* DH5α competent cells, and single colonies were picked for PCR identification and sequencing verification to obtain a positive recombinant editing vector.

[0028] (2) Agrobacterium-mediated genetic transformation of soybean The recombinant editing vector was introduced into wild-type soybean WS82 using Agrobacterium-mediated cotyledonary node transformation. The specific steps were as follows: Wild-type WS82 soybean seeds were sterilized and germinated under aseptic conditions; 5-7 day old cotyledonary nodes were used as explants; Agrobacterium-mediated transformation containing the recombinant editing vector was co-cultured with the explants for 3 days; the co-cultured explants were transferred to an induction medium containing selection markers for callus induction and shoot differentiation; when the differentiated shoots reached 3-5 cm in length, they were transferred to a rooting medium to induce rooting; after rooting, the seedlings were hardened off and transplanted into a greenhouse for cultivation to obtain transgenic soybean plants.

[0029] Example 2: Identification of disease resistance phenotypes between mutants and wild types Genomic DNA was extracted from transgenic soybean plants, and PCR amplification was performed using specific primers flanking the target sites. The amplified products were then sequenced for analysis. Plants showing base deletions, insertions, or substitutions were further subjected to homozygous self-pollination. After two consecutive generations of self-pollination and sequencing verification, the resulting transgenic soybean plants were successfully isolated. GmLNK2a , GmLNK2b , GmLNK2c , GmLNK2d A plant with homozygous mutations in all four genes is called a plant. gmlnk2-4m Four mutants.

[0030] wild-type WS82 and gmlnk2-4mSeeds of the four mutants were germinated for 2 days in a constant temperature incubator at 25℃. The germinated seeds were then planted in naturally occurring soybean root rot disease nurseries, where the soil contained abundant root rot pathogens (mainly Fusarium). Three replicate plots were set up for each material, with 50 plants per plot, spaced 20 cm × 30 cm apart. Field management followed conventional soybean cultivation techniques, including regular watering and weeding, without the application of fungicides.

[0031] When soybean plants entered the initial flowering stage, the physiological phenotypes of the two materials were statistically analyzed: Plant height measurement: the distance from the base of the plant to the growing point; Main root length measurement: the distance from the tip of the main root to the base of the root; Trifoliate leaf area measurement: Select the trifoliate leaves in the middle of the plant, take a picture, and use ImageJ to measure the area of ​​each trifoliate leaf.

[0032] Ten plants were randomly selected for each indicator for measurement. The mean and standard deviation were calculated, and the Student's test was used to analyze the significant differences.

[0033] Phenotypic results show that, compared with wild-type WS82, gmlnk2-4m All growth indicators of the four mutants were significantly reduced: Plant height measurement: The average plant height of wild-type WS82 was 43.7 cm. gmlnk2-4m The average plant height of the four mutants was 22.3 cm, which was 49% lower than that of the wild type. Figure 2 ); Taproot length measurement: The average taproot length of wild-type WS82 was 28.8 cm. gmlnk2-4m The average taproot length of the four mutants was 14 cm, which was 51.4% shorter than that of the wild type. Figure 3 Leaf area measurement of trifoliate compound leaves: The average leaf area of ​​trifoliate compound leaves of wild-type WS82 was 22.5 cm². gmlnk2-4m The tetramutant had an average leaf area of ​​13.1 cm² for its trifoliate compound leaves, which was 41.8% smaller than that of the wild type. Figure 4 ).

[0034] Since the test materials were planted in a root rot disease nursery, the above phenotypic differences in growth and development showed that there were highly significant differences between the two groups in all three phenotypic indicators. P <0.01), gmlnk2-4m The four mutants exhibit higher susceptibility to root rot pathogens in the diseased nursery, resulting in more severe infection and significantly inhibited plant growth. This result demonstrates that... GmLNK2This gene family plays a crucial positive regulatory role in soybean's resistance to root rot pathogens. Precise regulation of this gene family (such as overexpression) holds promise for developing new soybean varieties with enhanced resistance to root rot, offering significant potential for ensuring soybean yield and quality.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Knock out soybeans GmLNK2 Application of gene function in breeding disease-resistant soybean plants.

2. The application according to claim 1, characterized in that: The GmLNK2 Genes include GmLNK2a Gene, GmLNK2b Gene, GmLNK2c Genes and GmLNK2d Gene.

3. The application according to claim 2, characterized in that: The GmLNK2a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmLNK2b The nucleotide sequence of the gene is shown in SEQ ID NO.

2. GmLNK2c The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GmLNK2d The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

4. The application according to claim 3, characterized in that: The knockout is achieved through RNA interference technology or CRISPR / Cas9 gene editing technology; the disease resistance is resistance to soybean root rot.

5. A type of soybean GmLNK2 Gene tetra mutant gmlnk2-4m The construction method is characterized by, The steps are as follows: (1) Can be knocked out simultaneously GmLNK2a , GmLNK2b , GmLNK2c and GmLNK2d The three specific targets of the gene were constructed into the CRISPR / Cas9 gene editing vector; (2) The CRISPR / Cas9 gene editing vector was transferred into soybean plants using Agrobacterium-mediated transformation. After screening and sequencing verification, homozygous tetra mutant plants were obtained. gmlnk2-4m .

6. The soybean according to claim 5 GmLNK2 Gene tetra mutant gmlnk2-4m The construction method is characterized by: The GmLNK2a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmLNK2b The nucleotide sequence of the gene is shown in SEQ ID NO.

2. GmLNK2c The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GmLNK2d The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

7. The soybean according to claim 6 GmLNK2 Gene tetra mutant gmlnk2-4m The construction method is characterized by: The three specific targets are target 1, target 2 and target 3, with the sequence of target 1 as shown in SEQ ID NO.5, the sequence of target 2 as shown in SEQ ID NO.6 and the sequence of target 3 as shown in SEQ ID NO.

7.

8. The soybean according to claim 7 GmLNK2 Gene tetra mutant gmlnk2-4m The construction method is characterized by, The construction steps of the CRISPR / Cas9 gene editing vector are as follows: the vector is double-digested with an endonuclease, and the vector backbone is recovered; the specific target sequence is annealed to form double-stranded DNA and ligated to the vector backbone; the ligation product is transformed into E. coli, and single colonies are picked for PCR amplification and sequencing to obtain the gene editing vector.

9. The soybean according to claim 8 GmLNK2 Gene tetra mutant gmlnk2-4m The construction method is characterized by: The Agrobacterium-mediated method involves co-culturing Agrobacterium culture carrying gene-editing vectors with explants to induce callus tissue and shoot differentiation, followed by transfer to rooting medium to induce rooting, resulting in homozygous tetramutant plants. gmlnk2- 4m .

10. The application of the mutant constructed by the construction method according to any one of claims 5-9 in soybean disease resistance breeding, characterized in that: The disease resistance mentioned refers to resistance to soybean root rot.

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