Application of Glyma. 08g106300 gene in identifying saline-alkaline tolerance of soybeans

Abstract

The invention discloses application of a Glyma. 08g106300 gene in identifying saline-alkaline tolerance of soybeans, and relates to the technical field of biology. A FramCPU model is adopted to carry out GWAS analysis on 899 parts of soybean germplasm materials, the Glyma. 08g106300 gene related to the saline-alkaline tolerance of soybeans is obtained through screening, haplotype analysis and yeast transgene verification experiments prove that the mutation type of the Glyma. 08g106300 gene can really influence the saline-alkaline tolerance of the soybeans, and Hap1 haplotype soybean plants have higher saline-alkaline tolerance. The invention proves that the Glyma. 08g106300 gene is related to saline-alkaline tolerance of soybeans and can be used for identifying the saline-alkaline tolerance of the soybeans, so that a technical support is provided for breeding saline-alkaline tolerance varieties of the soybeans.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of the Glyma.08g106300 gene in identifying the salt and alkali tolerance of soybeans. Background Art

[0003] There is an urgent need to explore the salt and alkali tolerance genes of soybeans for application in the breeding of salt and alkali tolerant soybean varieties. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of the Glyma.08g106300 gene in identifying the salt and alkali tolerance of soybeans, so as to solve the problems existing in the above-mentioned prior art. The present invention has found through research that the Glyma.08g106300 gene is related to the salt and alkali tolerance of soybeans and can be used to identify the salt and alkali tolerance of soybeans, thereby providing technical support for the breeding of salt and alkali tolerant soybean varieties.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a KASP molecular marker for identifying the salt and alkali tolerance of soybeans, and the nucleotide sequence of the molecular marker is as shown in SEQ ID NO.9;

[0007] There is an SNP site at the 20th base of the molecular marker, which is a G / C mutation.

[0008] The present invention also provides a KASP primer combination for identifying the salt and alkali tolerance of soybeans, including two upstream primers with nucleotide sequences as shown in SEQ ID

[0009] NO.6 - 7 and a common downstream primer with a nucleotide sequence as shown in SEQ ID NO.8.

[0010] The present invention also provides the application of the above KASP primer combination in the preparation of a kit for identifying the salt and alkali tolerance of soybeans.

[0011] The present invention also provides a kit for identifying the salt and alkali tolerance of soybeans, including the above KASP primer combination.

[0012] The present invention also provides the application of the above KASP molecular marker, KASP primer combination or kit in identifying the salt and alkali tolerance of soybeans.

[0013] Further, when the above SNP site is the CC genotype, it indicates high salt and alkali tolerance of soybeans.

[0014] The present invention also provides a method for identifying the salt and alkali tolerance of soybeans, including the following steps:

[0015] Total RNA of the soybean sample to be detected was extracted and reverse transcribed into cDNA;

[0016] The above KASP primer combination was used to perform KASP PCR amplification on the cDNA, and the genotype was detected and the salt and alkali tolerance of soybeans was predicted: when the genotype is CC, it indicates that the salt and alkali tolerance of soybeans is high.

[0017] Furthermore, the reaction system for the KASP PCR amplification is as follows: 5 μL of template DNA, 0.14 μL of Primer mix, and 5 μL of KASP PCR MIX.

[0018] The present invention also provides the application of the salt-alkali-tolerant Glyma.08g106300 gene in improving the salt and alkali tolerance of soybeans. The nucleotide sequence of the salt-alkali-tolerant Glyma.08g106300 gene is as shown in SEQ ID NO.3;

[0019] The salt and alkali tolerance of soybeans is improved by overexpressing the salt-alkali-tolerant Glyma.08g106300 gene in soybean plants.

[0020] The present invention also provides the application of the salt-alkali-tolerant Glyma.08g106300 gene in improving the salt and alkali tolerance of yeast. The nucleotide sequence of the salt-alkali-tolerant Glyma.08g106300 gene is as shown in SEQ ID NO.3;

[0021] The salt and alkali tolerance of yeast is improved by overexpressing the salt-alkali-tolerant Glyma.08g106300 gene in yeast.

[0022] The present invention discloses the following technical effects:

[0023] The present invention used the FramCPU model to perform GWAS analysis on 899 soybean germplasm materials, screened out the Glyma.08g106300 gene related to the salt and alkali tolerance of soybeans, and through haplotype analysis and yeast transgenic verification experiments, it was confirmed that the mutation type of the Glyma.08g106300 gene can indeed affect the salt and alkali tolerance of soybeans, and soybean plants with the Hap1 haplotype have stronger salt and alkali tolerance.

[0024] The present invention proves that the Glyma.08g106300 gene is related to the salt and alkali tolerance of soybeans and can be applied to identify the salt and alkali tolerance of soybeans, thus providing technical support for the breeding of salt-alkali-tolerant soybean varieties. Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 is the identification result of the salt and alkali tolerance ability of soybean germplasm resources;

[0027] Figure 2 is the QQ plot (A) and Manhattan plot (B) of the association analysis of soybean salt and alkali tolerance traits at the germination stage;

[0028] Figure 3 is the haplotype analysis result of the Glyma.08g106300 gene; Hap2 is the sensitive haplotype, and Hap1 represents the salt and alkali tolerant haplotype;

[0029] Figure 4 is the amino acid sequence alignment result of the salt and alkali sensitive type and salt and alkali tolerant type Glyma.08g106300 proteins;

[0030] Figure 5 is the KASP genotyping result map of the Glyma.08G106300 gene;

[0031] Figure 6 is the transgenic yeast verification experiment result of Glyma.08g106300; among them, CK is the normal culture medium; SA is the salt and alkali stress culture medium. Detailed Embodiments

[0032] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0037] Example 1 Mining and Screening of Excellent Genes for Salt and Alkali Tolerance in Soybean

[0038] I. Mining Excellent Genes for Salt and Alkali Tolerance at the Bud Stage of Soybean by GWAS Analysis

[0039] Mining excellent genes for salt and alkali tolerance at the bud stage by GWAS analysis: The FarmCPU model was used to perform GWAS analysis on 899 materials (see Table 1), and the population structure was controlled by the first three components in the SNP principal component analysis (PCA) using the PLINKv1.90 software. The resulting P-values were adjusted at the α = 0.05 level using the Bonferroni correction, and the cut-off value was 9.44×10 -09 .

[0040] Table 1 Soybean Materials

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] After three repetitions, 899 soybean germplasm resources were identified for salt-alkali tolerance at the germination stage, and the results are shown in Figure 1 , where the average value of the salt-alkali stress index (the larger the value, the stronger the stress, and the smaller the value, the weaker the stress, the same below) was 36.15%, the median was 37.51%, the kurtosis was 0.179, and the skewness was -0.313. The results conform to the normal distribution and can be used for subsequent experimental analysis.

[0049] The FarmCPU model was used to perform GWAS analysis on salt-alkali tolerance at the germination stage of soybeans. At the threshold of P ≤ 1.75×10 -9 , the SNP with the highest effect value on chromosome 8 was detected as Gm08_8198079( Figure 2 ).

[0050] Haplotype analysis was performed on important candidate genes in the interval. In the gene Glyma.08g106300, a mutation occurred at 142 bp in the DNA segment, and the mutation of the base would cause changes in the amino acids of the protein (see Table 2 and Figure 4 ), which led to changes in the structure of the protein and then changes in the salt-alkali tolerance ability at the germination stage. The detection results of the salt-alkali stress index and haplotype analysis of soybean materials are shown in Figure 3 . The nucleotide sequence of the Glyma.08g106300 gene is shown in SEQ ID NO.1. Among them, the nucleotide sequence of the salt-sensitive Glyma.08g106300 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the salt-tolerant Glyma.08g106300 gene is shown in SEQ ID NO.3; the amino acid sequence of the salt-sensitive Glyma.08g106300 protein is shown in SEQ ID NO.4, and the amino acid sequence of the salt-tolerant Glyma.08g106300 protein is shown in SEQ ID NO.5.

[0051] Table 2 Sequence alignment of salt-tolerant and salt-sensitive Glyma.08g106300 genes and proteins

[0052] 142bp 291bp 309bp Salt-tolerant type (Hap1) C A G Salt-sensitive type (Hap2) G G A Amino acid change A>P / /

[0053] Note: " / " indicates that the amino acid has not changed.

[0054] The nucleotide sequence of the Glyma.08g106300 gene (SEQ ID NO.1):

[0055] ATGACGAGCAGCAAGAGGGTCCAAGACAGGAGCAAGAAGAAGAGGGTTCACGACCTCGAAGTCACAACCGAGAAATGGAAAATAGCCTCCAAAATCATCTACTTAATGGAGCTTCTCAAGCAAGAGCCCGAAATGGTCATT W CCGTTCGCTCCCTGGAGCACCACCGCAGACAAATCAACCTCCCCAAGCCCCACCGTGTCTCCGATTTCCTCCGCAAAACGCCCAACCTCTTCGAACTCTACAAGGACCAAAAGGGGGTGCTATGGTGCGGCATGACCTCAAAAGCCGA K AACTTGATGGAACAACA KCAAAGGGTCATCGAAGAACATGCCGACAAAGTCGCCGAACACGTCACGAGGTTTCTCATGATGTCGCTGGATAAAAGGCTTCCCTTGGAGAAGATTGCTCATTTCAGAAGAGATTTTGGGTTGCCGTTGGATTTTAGAGTCCATTGGGTGCATATGTATCCTCAGCATTTTAGGGTGGTGAAAGCACTTGATGGGGTTGAGTTTTTGGAGCTTGTGTCTTGGAACCCTGATTGGGCAATCACTGAGTTAGAGAAAAAGGTGGTGACAGAGAAAACTGCAACTACCAATACCCCAGGGATGCTTTCAATTCCATTTCCTTTGAAGTTCCCTGCGAATTATAAGAGGGTGTATCGTTACTATGGTGAAAAGATTCAGCATTTTCAGGAGATGTCTTATTTGTCTCCCTATGCTGATGCTCGTGGACTTAAGGCTGGATCATTGGAGTTTGATAAAAGGGCTGTTGCTGTTATGCATGAACTGCTTAGTTTCACCATTGAGAAGAGGTTGGTGACTGATCACCTCACACACTTTCGTTGGGAGCTGGTGATGCCCCAGAAGCTGATGAGGCTTCTGCTCAAGCATTGTGGGATCTTCTATGTGTCAGAGAGGGGGAAGAGGTTTAGTGTGTTCTTGACTGAGGCTTATGAGGGTTCTGAGCTGATTGAGAAATGCCCCTTGGTGCTGTGGAAGGAGAAGGTTTTGGGGCTTGTGGGGTATAGAGGGAGGAAGAAGAAGTTTGAGGTATGCAGTGATGATGGATCTGATGTGGAGGATCATGATGGTTTGGTTTGTGATCAGAGGGATTCTGAAGTGGGGGACTTGCATGTGCAGATTGAGCCTCGGGGTACCTTGGATTATGAGGATCCTTTACTTGGGGATGATTCTGAGATGGATGTTGGAGAGATTACATGA; Among them, the underlined positions are mutation sites, W is G or C, and K is A or G.

[0056] Nucleotide sequence of the salt-tolerant and alkali-tolerant Glyma.08g106300 gene (SEQ ID NO.2):

[0057] ATGACGAGCAGCAAGAGGGTCCAAGACAGGAGCAAGAAGAAGAGGGTTCACGACCTCGAAGTCACAACCGAGAAATGGAAAATAGCCTCCAAAATCATCTACTTAATGGAGCTTCTCAAGCAAGAGCCCGAAATGGTCATT C CCGTTCGCTCCCTGGAGCACCACCGCAGACAAATCAACCTCCCCAAGCCCCACCGTGTCTCCGATTTCCTCCGCAAAACGCCCAACCTCTTCGAACTCTACAAGGACCAAAAGGGGGTGCTATGGTGCGGCATGACCTCAAAAGCCGA A AACTTGATGGAACAACA GCAAAGGGTCATCGAAGAACATGCCGACAAAGTCGCCGAACACGTCACGAGGTTTCTCATGATGTCGCTGGATAAAAGGCTTCCCTTGGAGAAGATTGCTCATTTCAGAAGAGATTTTGGGTTGCCGTTGGATTTTAGAGTCCATTGGGTGCATATGTATCCTCAGCATTTTAGGGTGGTGAAAGCACTTGATGGGGTTGAGTTTTTGGAGCTTGTGTCTTGGAACCCTGATTGGGCAATCACTGAGTTAGAGAAAAAGGTGGTGACAGAGAAAACTGCAACTACCAATACCCCAGGGATGCTTTCAATTCCATTTCCTTTGAAGTTCCCTGCGAATTATAAGAGGGTGTATCGTTACTATGGTGAAAAGATTCAGCATTTTCAGGAGATGTCTTATTTGTCTCCCTATGCTGATGCTCGTGGACTTAAGGCTGGATCATTGGAGTTTGATAAAAGGGCTGTTGCTGTTATGCATGAACTGCTTAGTTTCACCATTGAGAAGAGGTTGGTGACTGATCACCTCACACACTTTCGTTGGGAGCTGGTGATGCCCCAGAAGCTGATGAGGCTTCTGCTCAAGCATTGTGGGATCTTCTATGTGTCAGAGAGGGGGAAGAGGTTTAGTGTGTTCTTGACTGAGGCTTATGAGGGTTCTGAGCTGATTGAGAAATGCCCCTTGGTGCTGTGGAAGGAGAAGGTTTTGGGGCTTGTGGGGTATAGAGGGAGGAAGAAGAAGTTTGAGGTATGCAGTGATGATGGATCTGATGTGGAGGATCATGATGGTTTGGTTTGTGATCAGAGGGATTCTGAAGTGGGGGACTTGCATGTGCAGATTGAGCCTCGGGGTACCTTGGATTATGAGGATCCTTTACTTGGGGATGATTCTGAGATGGATGTTGGAGAGATTACATGA; Among them, the underlined part is the mutation site.

[0058] Nucleotide sequence of the salinity-sensitive Glyma.08g106300 gene (SEQ ID NO.3):

[0059] ATGACGAGCAGCAAGAGGGTCCAAGACAGGAGCAAGAAGAAGAGGGTTCACGACCTCGAAGTCACAACCGAGAAATGGAAAATAGCCTCCAAAATCATCTACTTAATGGAGCTTCTCAAGCAAGAGCCCGAAATGGTCATT G CCGTTCGCTCCCTGGAGCACCACCGCAGACAAATCAACCTCCCCAAGCCCCACCGTGTCTCCGATTTCCTCCGCAAAACGCCCAACCTCTTCGAACTCTACAAGGACCAAAAGGGGGTGCTATGGTGCGGCATGACCTCAAAAGCCGA G AACTTGATGGAACAACA ACAAAGGGTCATCGAAGAACATGCCGACAAAGTCGCCGAACACGTCACGAGGTTTCTCATGATGTCGCTGGATAAAAGGCTTCCCTTGGAGAAGATTGCTCATTTCAGAAGAGATTTTGGGTTGCCGTTGGATTTTAGAGTCCATTGGGTGCATATGTATCCTCAGCATTTTAGGGTGGTGAAAGCACTTGATGGGGTTGAGTTTTTGGAGCTTGTGTCTTGGAACCCTGATTGGGCAATCACTGAGTTAGAGAAAAAGGTGGTGACAGAGAAAACTGCAACTACCAATACCCCAGGGATGCTTTCAATTCCATTTCCTTTGAAGTTCCCTGCGAATTATAAGAGGGTGTATCGTTACTATGGTGAAAAGATTCAGCATTTTCAGGAGATGTCTTATTTGTCTCCCTATGCTGATGCTCGTGGACTTAAGGCTGGATCATTGGAGTTTGATAAAAGGGCTGTTGCTGTTATGCATGAACTGCTTAGTTTCACCATTGAGAAGAGGTTGGTGACTGATCACCTCACACACTTTCGTTGGGAGCTGGTGATGCCCCAGAAGCTGATGAGGCTTCTGCTCAAGCATTGTGGGATCTTCTATGTGTCAGAGAGGGGGAAGAGGTTTAGTGTGTTCTTGACTGAGGCTTATGAGGGTTCTGAGCTGATTGAGAAATGCCCCTTGGTGCTGTGGAAGGAGAAGGTTTTGGGGCTTGTGGGGTATAGAGGGAGGAAGAAGAAGTTTGAGGTATGCAGTGATGATGGATCTGATGTGGAGGATCATGATGGTTTGGTTTGTGATCAGAGGGATTCTGAAGTGGGGGACTTGCATGTGCAGATTGAGCCTCGGGGTACCTTGGATTATGAGGATCCTTTACTTGGGGATGATTCTGAGATGGATGTTGGAGAGATTACATGA; Among them, the underlined part is the mutation site.

[0060] Amino acid sequence of salt-alkali sensitive Glyma.08g106300 protein (SEQ ID NO.4):

[0061] MTSSKRVQDRSKKKRVHDLEVTTEKWKIASKIIYLMELLKQEPEMVI A VRSLEHHRRQINLPKPHRVSDFLRKTPNLFELYKDQKGVLWCGMTSKAENLMEQQQRVIEEHADKVAEHVTRFLMMSLDKRLPLEKIAHFRRDFGLPLDFRVHWVHMYPQHFRVVKALDGVEFLELVSWNPDWAITELEKKVVTEKTATTNTPGMLSIPFPLKFPANYKRVYRYYGEKIQHFQEMSYLSPYADARGLKAGSLEFDKRAVAVMHELLSFTIEKRLVTDHLTHFRWELVMPQKLMRLLLKHCGIFYVSERGKRFSVFLTEAYEGSELIEKCPLVLWKEKVLGLVGYRGRKKKFEVCSDDGSDVEDHDGLVCDQRDSEVGDLHVQIEPRGTLDYEDPLLGDDSEMDVGEIT*。

[0062] Amino acid sequence of salt-tolerant Glyma.08g106300 protein (SEQ ID NO.5):

[0063] MTSSKRVQDRSKKKRVHDLEVTTEKWKIASKIIYLMELLKQEPEMVI P VRSLEHHRRQINLPKPHRVSDFLRKTPNLFELYKDQKGVLWCGMTSKAENLMEQQQRVIEEHADKVAEHVTRFLMMSLDKRLPLEKIAHFRRDFGLPLDFRVHWVHMYPQHFRVVKALDGVEFLELVSWNPDWAITELEKKVVTEKTATTNTPGMLSIPFPLKFPANYKRVYRYYGEKIQHFQEMSYLSPYADARGLKAGSLEFDKRAVAVMHELLSFTIEKRLVTDHLTHFRWELVMPQKLMRLLLKHCGIFYVSERGKRFSVFLTEAYEGSELIEKCPLVLWKEKVLGLVGYRGRKKKFEVCSDDGSDVEDHDGLVCDQRDSEVGDLHVQIEPRGTLDYEDPLLGDDSEMDVGEIT*。

[0064] II. Screening of Excellent Genes for Salt and Alkaline Tolerance at the Soybean Sprout Stage by KASP Markers

[0065] 1. Materials

[0066] 1.1 Soybean Plant DNA

[0067] A total of 96 DNA samples from salt and alkaline sensitive and tolerant soybean plants were extracted by the CTAB method.

[0068] 1.2 Main Reagents

[0069] The 2×Master Mix for ASPCR reagent was purchased from Chengdu Hanchen Guangyi Biotechnology Co., Ltd.

[0070] 1.3 Main Equipment and Instruments

[0071] High-speed low-temperature centrifuge, vortex shaker, water bath, nucleic acid and protein detector, fluorescence quantitative PCR amplifier, pipette, pipette tips and EP tubes, etc.

[0072] 2. KASP Primer Design

[0073] KASP primers were designed according to the SNP loci screened in the above process. It was required that the base sequences of the two parents were different at the SNP position, and there were no other SNPs within 20 bp upstream and 40 bp downstream of this SNP.

[0074] First, the CDS sequence was found using Phytozome. 20 bp upstream from the base where the SNP was located was used as the upstream primer F, and the base of the SNP was used as the 20th base of the left primer. Thus, there was the upstream primer F 1 , F 2 ; Using primer design software (primer3Plus.) or NCBI, fixing the upstream primer F, the common downstream primer R was obtained through blast, and the product size needed to be less than 60 bp; The left primers F 1 , F 2 were respectively added with adapters A 1 , A 2 (adapter sequence A 1 : GAAGGTGACCAAGTTCATGCT, A 2 : GAAGGTCGGAGTCAACGGATT), and the sequences F 1 A 1 , F 2 A 2 , R were synthesized. The primer sequences are shown in Table 2’.

[0075] Table 2’ KASP Primer Sequences

[0076] Primer Nucleotide sequence (5’-3’) Sequence number <![CDATA[F 1 A 1 > GAAGGTGACCAAGTTCATGCTAGAGCCCGAAATGGTCATTG SEQ ID NO.6 <![CDATA[F 2 A 2 > GAAGGTCGGAGTCAACGGATTAGAGCCCGAAATGGTCATTC SEQ ID NO.7 R GAAATCGGAGACACGGTGGG SEQ ID NO.8

[0077] 3. KASP PCR Detection and Results

[0078] Perform KASP PCR amplification using the KASP primers in Table 2’. The amplification system and procedure are shown in Tables 3 and 4.

[0079] KASP Molecular Marker:

[0080] AGAGCCCGAAATGGTCATT W CCGTTCGCTCCCTGGAGCACCACCGCAGACAAATC AACCTCCCCAAGCCCCACCGTGTCTCCGATTTC (SEQ ID NO.9), where W is G or C.

[0081] The genotyping results of the Glyma.08g106300 gene are shown in Figure 5 .

[0082] Table 3 KASP PCR System

[0083] Composition Volume Template DNA 5μL Primermix 0.14μL

[0084]

[0085] Table 4 KASP PCR Procedure

[0086]

[0087] Example 2 Functional Verification of the Glyma.08g106300 Gene

[0088] 1. Materials

[0089] 1.1 Strains and Vectors

[0090] The Escherichia coli strain DH5α was purchased from Beijing Tsingke Biotechnology Co., Ltd.; the yeast expression plasmid vector pYES2 was purchased from Beijing Coolaber Technology Co., Ltd.; the Saccharomyces cerevisiae INVScⅠ was purchased from Shanghai Angyu Biotechnology Co., Ltd.

[0091] 1.2 Main Reagents

[0092] The restriction endonucleases HindⅢ and EcoRⅠ were purchased from NEB (Beijing) Co., Ltd.; the homologous recombination enzyme was purchased from Nanjing Novoprotein Biotechnology Co., Ltd.; the RNA extraction kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; the reverse transcription kit was purchased from Yeasen Biotechnology Co., Ltd.; the product purification kit and plasmid extraction kit were purchased from Sangon Biotech Co., Ltd.; KOD-Fx high-fidelity enzyme, anhydrous ethanol, agarose, etc.

[0093] 1.3 Main Equipment and Instruments

[0094] High-speed low-temperature centrifuge, vortex shaker, water bath, autoclave, nucleic acid and protein detector, PCR amplifier, electrophoresis apparatus, gel imaging system, pipette, pipette tips, EP tubes, etc.

[0095] 2. Extraction and Detection of Total Plant RNA

[0096] Germinate soybean seeds and take the radicles after 5 days as samples. After sample collection, immediately freeze them in liquid nitrogen and transfer them to an ultra-low temperature refrigerator at -80 °C for storage. The RNA extraction method refers to the instruction manual of the RNA extraction kit. Finally, dissolve it in RNase-free water and detect the quality of the prepared samples.

[0097] First, measure the concentration of the extracted RNA and the OD 260 / 280 and OD 260 / 230 values, and then perform electrophoresis to detect the integrity of the RNA.

[0098] 3. Molecular Cloning of the Glyma08G.106300 Gene

[0099] 3.1 Synthesis of the First Strand of cDNA

[0100] Calculate the volume according to the RNA sample concentration, take 1 μg of total RNA as the template, and perform RT-PCR reverse transcription into cDNA with reference to the instruction manual of the reverse transcription kit.

[0101] 3.2 Cloning of the Glyma08G.106300 Gene and Recovery and Purification of PCR Products

[0102] 3.2.1 Primer Design

[0103] First, use Phytozome to find the CDS sequence, design primers using Snapgene, and send them to Shanghai Sangon for synthesis. The primer sequences are shown in Table 5:

[0104] Table 5 Primer Sequences for Cloning the Glyma08G.106300 Gene

[0105] Primer Sequence (5’-3’) Sequence number Glyma.08G106300_F tgatggatatctgcagaattcATGACGAGCAGCAAGAGGGTC SEQ ID NO.10 Glyma.08G106300_R actatagggaatattaagcttTGTAATCTCTCCAACATCCATCTCA SEQ ID NO.11

[0106] 3.2.2 Amplification of the Glyma08G.106300 Gene Fragment and Recovery and Purification of PCR Products

[0107] The PCR amplification system is shown in Table 6:

[0108] Table 6 PCR Amplification System

[0109] Composition Volume KOD-Fx high-fidelity enzyme 25μL Primer 3μL cDNA 2μL <![CDATA[ddH 2 O]]> 20μL

[0110] The PCR reaction procedure is as follows: pre-denaturation at 94°C for 5 min; denaturation at 98°C for 10 s, annealing at 55°C for 15 s; extension at 68°C for 10 s / kb, with 35 cycles set; extension at 68°C for 7 min. Verify the length of the PCR product by 1.0% agarose gel electrophoresis. Under ultraviolet light, use a newly unsealed blade to excise the single target DNA band as much as possible. Load the excised gel containing the DNA fragment into a pre-weighed 1.5 mL centrifuge tube, weigh it after loading, calculate the gel weight, and use a recovery kit for product recovery. Perform the experimental operations according to the instructions, and collect the DNA solution for storage at -20°C.

[0111] 3.3 Acquisition of pYES2 plasmid

[0112] The yeast expression vector used in this experiment is pYES2. Inoculate a small amount of purchased pYES2 plasmid on an LB solid medium (antibiotic: 100 mg / mL ampicillin) by streak plating and culture overnight. Then pick a single colony and culture it in a liquid medium by shaking overnight, and extract the pYES2 plasmid with reference to the plasmid extraction kit instructions.

[0113] To ligate the amplified target fragment to the vector, pYES2 plasmid and gene fragment with the same sticky ends need to be prepared. First, double-digest the pYES2 plasmid with EcorRⅠ and HindⅢ. After agarose gel electrophoresis, purify and recover the corresponding fragments. The digestion system is shown in Table 7:

[0114] Table 7 Digestion system

[0115]

[0116]

[0117] Add the system components to a 200 μL centrifuge tube, flick to mix evenly, briefly centrifuge for 5 s, then incubate at 37°C for 30 min and inactivate at 65°C for 20 min.

[0118] 3.4 Ligation and transformation of the target fragment and yeast expression vector

[0119] Measure the concentration of the corresponding fragments after recovery and purification, and use homologous recombinase to ligate the target fragment and the vector fragment to construct a recombinant plasmid vector. The ligation system is shown in Table 8:

[0120] Table 8 Ligation system

[0121] Composition Volume Homologous recombination enzyme 5μL Target fragment 1μL Linear vector 2μL <![CDATA[ddH 2 O]]> 2μL

[0122] Add the system components to a 200 μL centrifuge tube, flick to mix evenly, briefly centrifuge for 5 s, then place at 50°C for 15 min.

[0123] The recombinant plasmid vector was transferred into DH5α competent cells, and the antibiotic in the medium was 100 mg / mL ampicillin. After overnight culture, the colonies growing on the plate were ampicillin-resistant bacteria after transformation. Single colonies were picked and inoculated into a liquid medium containing ampicillin, and cultured overnight at 37 °C with 220 rpm. Using the overnight cultured bacterial solution as a template, bacterial solution PCR was performed. The positive bacterial solution identified by PCR was selected, and the recombinant plasmid vector was extracted using a plasmid extraction kit.

[0124] 3.5 Yeast transformation

[0125] Take freshly prepared yeast competent cells, and refer to the transformation section of the instruction manual to perform yeast transformation at room temperature. Finally, take 50 μL of the transformation mixture and evenly coat it on an SD-Ura (glucose) solid medium, and place it in an incubator at 30 °C for 2 - 4 days until white colonies grow.

[0126] Pick the growing white single colonies into 5 mL of SD-Ura liquid medium, culture overnight at 28 °C with 180 rpm, take the bacterial solution and perform cell disruption treatment at 100 °C for 5 min, and use 5 μL of the disrupted bacterial solution as a template for PCR detection to determine whether the target gene is accurately integrated into the yeast expression vector.

[0127] 3.6 Phenotypic identification of recombinant yeast

[0128] The recombinant yeasts were respectively renamed INVScⅠ(pYES2-Glyma08G.106300 Hap1 ) and INVScⅠ(pYES2-Glyma08G.106300 Hap2 ); among them, INVScⅠ(pYES2-Glyma08G.106300 Hap1 ) was the salt-tolerant Glyma.08g106300 gene imported in Example 1; NVScⅠ(pYES2-Glyma08G.106300 Hap2 ) was the salt-sensitive Glyma.08g106300 gene imported in Example 1.

[0129] To verify the effect of salt stress on transgenic yeast, a method of mixed salt stress with NaCl, Na 2 CO 3 , NaHCO 3 and Na 2 SO 4 (molar ratio 1:1:9:9) was used to observe its growth status, and the experiment was repeated three times.

[0130] (1) In a laminar flow hood, pick the recombinant yeast with successful verification and the yeast transfected with empty vector into SD-Ura liquid medium (glucose), and culture them with shaking at 28 °C and 180 rpm for 24 h;

[0131] (2) Centrifuge at 4000 rpm for 1 min, add 10 mL of induction medium with galactose (SD-Ura), adjust the OD 600 value to 0.4, and culture with shaking at 30 °C and 180 rpm for 36 h;

[0132] (3) Centrifuge at 4000 rpm for 1 min;

[0133] (4) Resuspend the cells with sterile water, measure the OD of the cell suspension 600 , and uniformly adjust the OD 600 to 1.8;

[0134] (5) Uniformly dilute the cell suspension by 10 -1 times, 10 -2 times, 10 -3 times, 10 -4 times;

[0135] (6) Using the INVScⅠ (pYES2) yeast transfected with empty vector as a control, take 3 μL of the undiluted cell suspension and the diluted cell suspension respectively, and inoculate them on normal YPDA solid medium and YPDA solid medium with mixed saline-alkali stress;

[0136] (7) Culture at 28 °C for 5 d;

[0137] (8) Observe and record the growth of the plates, and analyze and compare the differences in yeast growth.

[0138] 4. Results

[0139] In this example, a transgenic verification experiment was carried out in yeast to verify the function of Glyma.08g106300. The results showed that the transgenic yeast salt-tolerant haplotype (N: Glyma.08g106300 Hap1 ) developed salt-tolerant ability in the salt-tolerant medium; while the transgenic yeast sensitive haplotype (M: Glyma.08g106300 Hap2 ) did not develop salt-tolerant ability in the salt-tolerant medium ( Figure 6 ).

[0140] In summary, the above experimental results fully illustrate that the mutation type of the Glyma.08g106300 gene can affect the salt-tolerant ability of soybean at the germination stage, and the soybean plants with the Hap1 haplotype have stronger salt-tolerant ability.

[0141] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A KASP molecular marker for identifying soybean salt-alkali tolerance, characterized in that: The nucleotide sequence of the molecular marker is shown in SEQ ID NO.9; There is a SNP site at the 20th base of the molecular marker, which is a G / C mutation.

2. A KASP primer combination for identifying soybean salt-alkali tolerance, characterized in that: It includes two upstream primers with nucleotide sequences as shown in SEQ ID NO.6-7 and a universal downstream primer with a nucleotide sequence as shown in SEQ ID NO.

8.

3. Use of the KASP primer combination as claimed in claim 2 in preparing a kit for identifying the salt-alkali tolerance of soybean.

4. A kit for identifying the salt-alkali tolerance of soybeans, characterized in that: Comprising the KASP primer combination described in claim 2.

5. Use of the KASP molecular marker according to claim 1, the KASP primer combination according to claim 2 or the kit according to claim 4 in identifying the salt-alkali tolerance of soybean.

6. The use according to claim 5, characterized in that: When the SNP site described in claim 1 is of CC genotype, it indicates that the soybean has high salt-alkali tolerance.

7. A method for identifying the salt-alkali tolerance of soybeans, characterized in that: The following steps are involved: The total RNA of the soybean sample to be tested is extracted and reverse transcribed to obtain cDNA; The cDNA is amplified by KASP PCR using the KASP primer combination described in claim 2 to detect the genotype and predict the salt-alkali tolerance of soybean: when the genotype is CC, it indicates that the salt-alkali tolerance of soybean is high.

8. The method according to claim 7, characterized in that: The reaction system of the KASP PCR amplification is: 5 μL of template DNA, 0.14 μL of Primer mix and 5 μL of KASPPCRMIX.

9. Application of the salt-alkali tolerant Glyma.08g106300 gene in improving the salt-alkali tolerance of soybean, characterized in that: The nucleotide sequence of the salt-alkali tolerant Glyma.08g106300 gene is as shown in SEQ ID NO.3; The salt-alkali tolerance of soybean plants is improved by overexpressing the salt-alkali tolerant Glyma.08g106300 gene in the soybean plants.

10. Application of salt-alkali tolerant Glyma.08g106300 gene in improving salt-alkali tolerance of yeast, characterized in that: The nucleotide sequence of the salt-alkali tolerant Glyma.08g106300 gene is as shown in SEQ ID NO.3; The salt-alkali resistance of yeast is improved by overexpressing the salt-alkali resistance Glyma.08g106300 gene in yeast.

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