A method for enhancing the ability of soybean to resist southern soybean leaf enation virus by gene editing and application thereof

Abstract

The present application relates to the field of molecular biology and genetic engineering technology, and particularly relates to a method for enhancing the ability of soybean to resist southern soybean leaf crinkle disease through gene editing and application thereof, comprising: using CRISPR / Cas9 gene editing technology to target edit the nucleotide sequence of a soybean gene GLYMA_12G233000, The nucleotide sequence of the gene GLYMA_12G233000 is SEQ ID NO: 1. The present application screens GLYMA_ 12G233000 As a target gene, the existing soybean variety sensitive to SSCLD is genetically modified using gene editing technology, which can realize efficient, rapid and specific target editing and knockout of the soybean gene, change the expression of the gene GLYMA_12G233000 After gene editing, the leaf crinkle symptom of the soybean plant disappears, the ability to resist leaf crinkle disease is significantly enhanced, the resistance of the soybean variety (line) to southern soybean leaf crinkle disease is rapidly improved, and a non-transgenic soybean plant can be obtained, which greatly improves the breeding progress of soybean varieties resistant to southern soybean leaf crinkle disease.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and genetic engineering, and in particular to a method for enhancing the resistance of soybeans to southern soybean wrinkling disease through gene editing and its application. Background Technology

[0002] Soybeans are an important grain, oilseed, and feed crop in my country, playing a vital role in the country's national economic development. Southern soybean producing areas are among my country's major high-protein soybean production regions, providing high-quality raw materials for soybean protein processing. In recent years, a leaf-wrinkling disease similar to, but not caused by, soybean virus, has appeared in soybean production in Guangxi, Guangdong, Fujian, and Guizhou provinces. This disease is called Southern Soybean Crinkle Leaf Disease (SSCLD). Research indicates that SSCLD in these regions is caused by a certain pathogenic factor in the southern soil. In severe cases, it can lead to a soybean yield reduction of approximately 40%, posing a potential safety risk to soybean production in southern China.

[0003] SSCLD requires both a pathogenic factor and the gene controlling SSCLD in soybeans to be present for soybeans to exhibit leaf wrinkling symptoms. However, the nature of the pathogenic factor is currently unknown, making it difficult to artificially control it and screen for SSCLD-resistant germplasm. Therefore, without molecular markers or a pathogenic factor environment, breeders find it difficult to selectively breed varieties resistant to southern soybean leaf wrinkling. Consequently, selected soybean varieties may carry the gene controlling SSCLD, exhibiting leaf wrinkling and affecting yield in environments with the pathogenic factor. In actual production, it is common to encounter varieties that perform well in the absence of the pathogenic factor, but experience severe leaf wrinkling and yield reduction in areas where the pathogenic factor is present.

[0004] The applicant of this invention previously established field identification methods for SSCLD (Chen Wenjie et al., A method for identifying the occurrence characteristics and symptomatic level of soybean leaf wrinkling, 2020, Soybean Science, 39(3): 431-441) and molecular marker-assisted identification methods (Chen Wenjie et al., Development of molecular markers for the auxiliary identification of southern soybean leaf wrinkling based on transcriptome sequencing, 2023, Southern Agricultural Journal), providing technical reference for the rapid diagnosis and treatment of SSCLD in soybean production. However, how to enhance the ability of soybean to resist southern soybean leaf wrinkling still needs in-depth research. Currently, there are many publicly available candidate genes for controlling southern soybean leaf wrinkling, such as GLYMA_18G081800, GLYMA_08G325400, GLYMA_19G207100, GLYMA_12G223100, etc. However, no one has yet verified whether these candidate genes control SSCLD, nor has anyone screened and utilized these candidate genes to breed new varieties resistant to southern soybean wrinkling disease. Moreover, the current breeding of new varieties mainly involves introducing the target gene into other soybean varieties through hybridization and backcrossing, which is a long-term, labor-intensive, and costly breeding method. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a method for enhancing soybean resistance to Southern Soybean Wrinkled Leaf Disease through gene editing and its application. This method rapidly improves soybean resistance to Southern Soybean Wrinkled Leaf Disease through gene editing, yielding non-transgenic soybean plants and significantly accelerating the breeding process for soybean varieties resistant to Southern Soybean Wrinkled Leaf Disease. The specific technical solution is as follows: A method for enhancing soybean resistance to southern soybean wrinkling disease through gene editing includes: targeting and editing soybean genes using gene editing (CRISPR / Cas9) technology. GLYMA_12G233000 The gene GLYMA_12G233000 The nucleotide sequence is SEQ ID NO:1.

[0006] Preferably, the above method includes selecting soybean genes. GLYMA_12G233000 The target sequence SEQ2, wherein the target sequence SEQ2 is a soybean gene GLYMA_12G233000 The target sequence is a fragment of the 5'UTR region from bases 93 to 107 and the first CDS from bases 1 to 5, and the nucleotide sequence of the target sequence is shown in SEQ ID NO: 2.

[0007] Preferably, in the above method, CRISPR / Cas9 gene editing is used to target and knock out soybean genes. GLYMA_12G233000 .

[0008] Preferably, in the above method, the soybean gene is knocked out using CRISPR / Cas9 gene editing. GLYMA_12G233000 The 97th to 99th base sequence of the 5'UTR region.

[0009] Preferably, in the above method, the CRISPR / Cas9 gene editing system includes a gRNA scaffold, the nucleotide sequence of which is shown in SEQ ID NO: 3.

[0010] Preferably, the above method specifically includes the following steps: (1) Selection of soybean genes GLYMA_12G233000 The target sequence SEQ2 was determined, and primer P233000 (SEQ5) was designed to detect gene editing targets. The specific sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6. (2) Construct a CRISPR / CAS9 overexpression vector and edit the target gene; (3) Soybean genetic transformation was carried out using the cotyledon method to obtain transgenic plants; the transgenic plants were identified to obtain transgenic positive seedlings; (4) T0 transgenic seeds harvested from mature transgenic positive seedlings were planted, and RBA protein test strips were used for rapid detection and target site sequencing to screen single-plant lines that were successfully edited at the target site and were non-transgenic.

[0011] Preferably, in the above method, step (2) specifically includes: synthesizing the target sequence SEQ2; obtaining the promoter Gm-U6 fragment SEQ4 and the gRNAscaffold fragment SEQ3 from the T vector by PCR; ligating SEQ4, SEQ2 and SEQ3 by bridge PCR to obtain the target fragment; linearizing the CAS9 vector; then performing a reaction to ligate the target fragment to the CAS9 vector to obtain the gene editing expression vector; infecting the wrinkled soybean variety GY_C using the cotyledon infection method, culturing, extracting DNA, and identifying the effectiveness of the gene editing vector by sequence comparison detection.

[0012] Preferably, in the above method, step (3) specifically includes: transferring the expression vector into Agrobacterium tumefaciens, using Agrobacterium tumefaciens-mediated transformation to genetically transform the wrinkled soybean line, and obtaining transgenic plants through infection, explant induction, shoot induction, and rooting induction; identifying transgenic positive seedlings by Basta smear detection and RBA protein strip rapid detection methods.

[0013] Preferably, in the above method, the single-plant strain is in GLYMA_12G233000 Three bases from 97 to 99 in the 5'UTR region were knocked out.

[0014] On the other hand, the present invention provides an application of the above-described method in regulating soybean resistance to southern soybean leaf wrinkling disease.

[0015] Compared with the prior art, the beneficial effects of the present invention are: This invention screens GLYMA_12G233000 Using gene editing technology to genetically modify existing SSCLD-sensitive soybean varieties (lines) as target genes can achieve efficient, rapid, and specific modification of soybeans. GLYMA_12G233000 Genes are targeted for editing and knockout, altering... GLYMA_12G233000 The expression of the gene-edited soybean plant eliminated the leaf wrinkling symptoms and significantly enhanced its resistance to leaf wrinkling disease. This rapidly improved the resistance of soybean varieties (lines) to leaf wrinkling disease in southern soybeans and produced non-transgenic soybean plants. This greatly accelerated the breeding progress of soybean varieties resistant to leaf wrinkling disease in southern soybeans and significantly shortened the breeding cycle, providing methodological support for the targeted improvement of soybean varieties resistant to SSCLD. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are 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 This is a BSA-seq localization result diagram of the F2 population of GC8 and Y2017-1 in an embodiment of the present invention; Figure 2 The wrinkled leaf gene in this embodiment of the invention CL12 Detailed positioning result image; Figure 3 Embodiments of the present invention GLYMA_12G233000 Gene editing target selection; Figure 4 Comparison of target sequences of GY_C, the successfully edited gene line GY_C-CL12-CR6, and the negative control GY_C in this embodiment of the invention; Figure 5 In the GYC_ wrinkled leaf environment of this embodiment of the invention CL12 - Comparison of the wrinkled leaf appearance of CR6 and the negative control GY_C. Detailed Implementation

[0018] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0019] The tested varieties were Guichun 8 (GC8, normal leaves, leaf wrinkling grade 0) and Yuechun 2017-1 (Y2017-1, wrinkled leaves, leaf wrinkling grade 4). The soybean line GY_C (leaf wrinkling grade 4) was a heterozygous line derived from the sexual hybridization of Guichun 8 and Yuechun 2017-1. For details, please refer to the literature (Chen Wenjie et al., leaf morphological changes and their effects on yield traits when leaf wrinkling occurs in soybeans in southern China, Journal of Southern Agriculture, 2022, 53(2): 460-468).

[0020] Example A method for enhancing soybean resistance to southern soybean leaf wrinkling disease through gene editing includes the following steps: (1) An F2 population was constructed using the normal leaf material GC8 and the wrinkled leaf material Y2017-1, and the candidate genes controlling soybean wrinkling disease in southern China were identified using BSA-seq. CL12 Located to soybean chromosome 12 (see Figure 1 ), and develop primers for candidate regions, utilizing F2, BC1F2, and F 2:5 , BC8F2, BC9F2, BC 10 Genotyping of F2 and other progeny individuals narrowed the candidate gene range to 56.07 kb within the range of 40609871-40665938 bp (see [link to source genotype]). Figure 2 There are 13 candidate genes in total (Table 1). Based on gene function annotation information and candidate gene qPCR results, [the following genes were selected]. GLYMA_12G233000 As a candidate gene for controlling SSCLD, a literature search shows that there are currently no reports verifying that this gene controls SSCLD.

[0021] Table 1 Functional information of genes within candidate regions (2) sgRNA target sequence design: The longest transcribed version of this gene, SEQ1 (SEQ ID NO:7), was downloaded from the Williams82 a4 reference genome; it was then processed using the online gene editing design website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). GLYMA_12G233000 A 20bp target sequence SEQ2 was designed at the 5' UTR. SEQ2 has a PAM sequence at the 5' end of the reference genome. Figure 3The selection of CRISPR / CAS9 target sequences was assessed; SEQ2 simultaneously met the requirements for specificity and conservation. Specificity met the gene editing target specificity requirement; conserved fragments were screened using the SNP variant site analysis database (https: / / yanglab.hzau.edu.cn / SoyMD / # / variation), and conservation met the gene editing needs of most soybean germplasm. Primers P233000 (SEQ5) were designed to detect gene editing targets, including the forward primer sequence SEQ ID NO:5 and the reverse primer sequence SEQ ID NO:6.

[0022] SEQ2: ATCATGGTCCTGCTGGTGT (SEQ ID NO: 2); (3) Construction of gene editing vectors: The target sequence SEQ2 was synthesized. Using a T vector containing the promoter Gm-U6 and gRNA scaffold sequence as a template, the promoter Gm-U6 fragment SEQ4 and gRNA scaffold fragment SEQ3 were obtained by PCR amplification. The target fragment was obtained by bridging PCR by linking SEQ4, SEQ2 and SEQ3. The bridging PCR reaction system is shown in Table 2.

[0023] SEQ3: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (SEQ ID NO: 3) SEQ4: AAAATAAATGGTAAAATGTCAAATCAAAACTAGGCTGCAGTATGCAGAGCAGAGTCATGATGATACTACTTACTACACCGATTCTTGTGTGCAGAAAAATATGTTAAAATAATTGAATCTTTCTCTAGCCAAATTTGACAACAATGTACACCGT TCATATTGAGAGACGATGCTTCTTGTTTGCTTTCGGTGGAAGCTGCATATACTCAACATTACTCCTTCAGCGAGTTTTCCAACTGAGTCCCACATTGCCCAGACCTAACACGGTATTCTTGTTTAATGAAATGTGCCACCACATGGATT (SEQ ID NO:4) Table 2 Fusion PCR Reaction System The CAS9 vector (0645 vector with kanamycin resistance) was linearized according to the enzyme digestion reaction system (see Table 3) (37℃ water bath for 30 min); then the target fragment was ligated into the CAS9 vector according to the reaction system (see Table 4) (50℃ reaction for 30 min) to obtain the target vector; subsequently, the ligated plasmid was transformed into Escherichia coli DH5α for cloning and proliferation, single colonies were picked, and positive single colonies were identified by PCR for later use.

[0024] Table 3. CAS9 vector linearization enzymatic digestion reaction system Table 4 Connection Reaction System (4) Validation of gene editing vectors: The target vector was transformed into *E. coli* for cloning and propagation, and plasmids were extracted using the alkaline lysis method. *Agrobacterium tumefaciens* K599 competent cells were transformed using electroporation. *Agrobacterium* colonies were identified by PCR. After successful PCR identification of positive clones, *Agrobacterium*-mediated cotyledon infection was used to infect the wrinkled-leaf soybean variety GY_C (sensitive to southern soybean wrinkling disease) for 30 min. The cells were then placed in culture dishes for rooting for 2 weeks. After hairy roots grew from most cotyledon nodes, the hairy roots were harvested, and DNA was extracted from the relevant tissues using the CTAB method. The target sequence fragment corresponding to primer P233000 was amplified by PCR and sent to the company for sequencing. The gene editing effectiveness was detected by sequence comparison. The primer P233000 sequence (SEQ5) was used for sequencing to detect the editing site.

[0025] SEQ5: P233000F: 5'-TACGTCAGATATTCTTTCCCTCAAAC-3' (SEQ ID NO: 5); P233000R: 5'-TGTAAACGGAAGAAGCAGGGAAC-3' (SEQ ID NO: 6).

[0026] (5) Soybean genetic transformation using the cotyledon method: After the gene editing vector was confirmed to be effective by hairy root testing, E. coli plasmids were extracted by alkaline cleavage and transformed into Agrobacterium EHA105 by electroporation. The Agrobacterium-mediated transformation method for soybean optimized by Li et al. (Optimization of agrobacterium-mediated transformation in soybean, Frontiers in Plant Science) was used to genetically transform the recipient material GY_C. After infection-induced explant-bud induction-rooting induction, transgenic plants were obtained. (6) Screening of positive transformants: Positive transgenic seedlings were identified using methods such as Basta smear test and rapid RBA protein test strip detection. (7) Screening of gene-edited strains: Positive transgenic seedlings were harvested to obtain T0 generation transgenic plant seeds. These seeds were planted, and positive transgenic plants were rapidly detected using RBA protein strips. Gene editing effectiveness was assessed using P233000 primer sequencing at the target site. Single plants successfully edited at the target site and not transgenic were selected. Through propagation of these single plants, the successfully edited GLYMA_12G233000 gene editing line GY_C-CL12-CR6 was obtained. This line... GLYMA_12G233000 Three bases were successfully deleted from the 5'UTR region. See details. Figure 4 .

[0027] In a wrinkled leaf environment, compared to the negative control GY_C, the leaves of the edited strain GY_C-CL12-CR6 became normal (see...). Figure 5 The negative control GY_C showed wrinkled leaves, indicating that the wrinkled leaf symptoms disappeared in the gene-edited soybean plants, and their resistance to wrinkled leaves was significantly enhanced. Furthermore, a field planting comparison experiment showed that the field traits of the gene-edited soybean plants were not significantly different from those of the negative control; data are detailed in Table 5.

[0028] Table 5. Field phenotypic performance of gene-edited and wild-type lines at spring sowing in 2025. In summary, this invention utilizes gene editing technology in soybeans... GLYMA_12G233000 The target site for editing was identified in the gene, primers were designed based on the target site sequence, a CRISPR / Cas9 vector was constructed, soybean tissue was transformed using Agrobacterium-mediated transformation, and the resulting product was screened and identified. GLYMA_12G233000 A genetically modified, non-transgenic soybean line, GY_C-CL12-CR6, resistant to southern soybean wrinkling disease, was developed. This method, applied to the breeding of soybean lines resistant to southern soybean wrinkling disease, eliminates the need for hybridization and significantly shortens the soybean variety breeding cycle.

[0029] SEQ1: GLYMA_12G233000 The longest transcribed version of the gene (SEQ ID NO:7) consists of the first 1-107 positions forming the 5'UTR region, the last 260 positions (indicated by the yellow part) forming the 3'UTR region, and the middle part is the CDS region.

[0030] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for enhancing the resistance of soybeans to soybean leaf wrinkling disease in southern China through gene editing, characterized in that, include: Targeted editing of soybean genes using gene editing technology GLYMA_12G233000 The gene GLYMA_12G233000 The nucleotide sequence is SEQ ID NO:1; soybean gene was selected. GLYMA_12G233000 The target sequence SEQ2, whose nucleotide sequence is shown in SEQ ID NO: 2, is used to target and knock out the soybean gene using CRISPR / Cas9 gene editing. GLYMA_12G233000 The 97th to 99th base sequence of the 5'UTR region of the longest transcribed version sequence, which is shown in SEQ ID NO:

7.

2. The method according to claim 1, characterized in that, The CRISPR / Cas9 gene editing includes gRNA scaffold, the nucleotide sequence of which is shown in SEQ ID NO:

3.

3. The method according to claim 1, characterized in that, Specifically, the following steps are included: (1) Selection of soybean genes GLYMA_12G233000 The target sequence SEQ2 was determined, and primer P233000 was designed to detect the gene editing target. (2) Construct a CRISPR / CAS9 overexpression vector and edit the target gene; (3) Soybean genetic transformation was carried out using the cotyledon method to obtain transgenic plants; the transgenic plants were identified to obtain transgenic positive seedlings; (4) T0 transgenic seeds harvested from mature transgenic positive seedlings were planted, and BAR protein test strips were used for rapid detection and target site sequencing to screen single-plant lines that were successfully edited at the target site and were non-transgenic.

4. The method according to claim 3, characterized in that, Step (2) specifically includes: synthesizing the target sequence SEQ2; obtaining the promoter Gm-U6 fragment SEQ4 and the gRNA scaffold fragment SEQ3 from the T vector by PCR; ligating SEQ4, SEQ2 and SEQ3 by bridge PCR to obtain the target fragment; linearizing the CAS9 vector; then performing a reaction to ligate the target fragment into the CAS9 vector to obtain the gene editing expression vector; infecting the wrinkled soybean variety using the cotyledon infection method, culturing, extracting DNA, and identifying the effectiveness of the gene editing vector by sequence comparison detection.

5. The method according to claim 3, characterized in that, The specific steps (3) include: transferring the expression vector into Agrobacterium tumefaciens, using Agrobacterium tumefaciens-mediated transformation to genetically transform the wrinkled soybean line, and obtaining transgenic plants through infection, explant induction, shoot induction, and rooting induction; and identifying transgenic positive seedlings by Basta smear detection and BAR protein test strip rapid detection methods.

6. The application of the method as described in any one of claims 1 to 5 in enhancing the resistance of soybean to southern soybean leaf wrinkling disease.

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