Soybean ethylene synthesis key gene mutant NKea-1 as well as preparation method and application thereof

Abstract

The invention relates to the technical field of plant biology, in particular to a soybean ethylene synthesis key gene mutant NKea-1 and a preparation method and application thereof, the key gene is located in the 460822-463208 base interval of soybean Gm01 chromosome DNA, the mutant NKea-1 is added with a T base behind the 462036th base in the interval, the nucleotide sequence of the mutant gene is as shown in SEQ ID NO.2, the nucleotide sequence of the mutant NKea-1 is as shown in SEQ ID NO.1, and the nucleotide sequence of the mutant NKea-1 is as shown in SEQ ID NO.2. The amino acid sequence of the protein coded by the mutant gene coding region in the mutant NKea-1 is as shown in SEQ ID NO. 4. According to the invention, the soybean ethylene synthesis key gene mutant NKea-1 is obtained by utilizing a CRISPR / Cas9 gene editing technology, the alkali stress resistance of the mutant is greatly improved, and the mutant can be used as a parent material to be used for breeding a new variety of alkali-resistant or stress-resistant high-quality soybean with other excellent characters through hybridization or molecular breeding and the like.

Description

Technical Field

[0001] The present invention relates to the field of plant biotechnology, in particular to a mutant of a key gene for soybean ethylene synthesis NKea-1 , its preparation method and application. Background Art

[0002] Soil salinization is a major threat to the sustainable development of global agriculture. The global saline-alkali land area is about 950 million hectares, and the total saline-alkali land area in my country is 115 million mu, of which the available saline-alkali arable land resources account for 25%. + 、Cl - Multiple mechanisms such as imbalance of nutrients, oxidative stress and high pH inhibit plant growth, leading to reduced crop yields or even crop failure.

[0003] Breeding salt-alkali-tolerant crops is a key component of the comprehensive development and utilization of saline-alkali land. Soybeans, as a major economic crop that serves both food and oil as well as feed, are also a fundamental and strategic resource crucial to national economy and livelihoods, as well as food security. Uncovering soybean salt-alkali-tolerant genes and conducting in-depth research into the functions and mechanisms of action of key genes is not only crucial for applying genetic engineering techniques to cultivate salt-alkali-tolerant transgenic soybeans and alleviating the crisis facing my country's soybean industry, but also provides a valuable reference for the breeding of other salt-alkali-tolerant crops, and holds even greater significance for the full utilization of saline-alkali land and the sustainable development of agriculture.

[0004] Ethylene is a gaseous hormone with a very simple chemical structure. It is one of the oldest plant hormones and plays a crucial role in plant growth, development, and defense responses. Previous studies in the model plant Arabidopsis thaliana have shown that different abiotic stresses can affect the expression of different genes involved in key enzymes in ethylene synthesis.

[0005] However, there are currently no reports on the response of soybean ethylene synthesis-related genes to saline-alkali stress, nor are there any studies on how to use soybean ethylene synthesis-related genes for breeding. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-1 The second object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-1 The third object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-1 application.

[0007] In order to achieve the first purpose, the technical solution adopted by the present invention is: A mutant of a key gene for soybean ethylene synthesis NKea-1The key gene for soybean ethylene synthesis is located in the 460822-463208 base interval of soybean chromosome DNA Gm01. NKea-1 The nucleotide sequence of the mutant gene is shown in SEQ ID NO.2.

[0008] Furthermore, the mutant NKea-1 The amino acid sequence of the protein encoded by the coding region of the mutant gene is shown in SEQ ID NO.4.

[0009] In order to achieve the second purpose, the technical solution adopted by the present invention is: A mutant of a key gene for soybean ethylene synthesis NKea-1 A preparation method for preparing any of the above soybean ethylene synthesis key gene mutants NKea-1, The steps include: S100, using CRISPR / Cas9 technology to construct mutants NKea-1 Gene editing vectors; S200, introduce gene editing vectors into plant cells through Agrobacterium-mediated plant transformation technology to screen mutants NKea-1 .

[0010] Furthermore, step S100 includes the following steps: S110, using pCBC-DT1T2 as template, design primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR; S120, amplify the sgRNA1 sequence using DT1-BsF as the forward primer and DT1-F0 as the reverse primer to obtain U6-26p-sgRNA1-sgRNA-Sc-U6-26t; The sgRNA2 sequence was amplified using DT2-R0 as the forward primer and DT2-BsR as the reverse primer to obtain U6-29p-sgRNA2-sgRNA-Sc-U6-29t; S130, U6-26p-sgRNA1-sgRNA-Sc-U6-26t and U6-29p-sgRNA2-sgRNA-Sc-U6-29t were evenly mixed, and amplified and synthesized using primers DT1-BsF and DT2-BsR to obtain sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2-(sgRNA-Sc); S140. Add BsaI enzyme, T4 ligase, and pBSE401 vector to sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2-(sgRNA-Sc) to perform assembly reaction to obtain mutants. NKea-1gene editing vectors.

[0011] Furthermore, in step S110, the nucleotide sequence of primer DT1-BsF is shown as SEQ ID NO.5, the nucleotide sequence of primer DT1-F0 is shown as SEQ ID NO.6, the nucleotide sequence of primer DT2-R0 is shown as SEQ ID NO.7, and the nucleotide sequence of primer DT2-BsR is shown as SEQ ID NO.8.

[0012] In order to achieve the third purpose, the technical solution adopted by the present invention is: A mutant of a key gene for soybean ethylene synthesis NKea-1 Application, such as any of the above soybean ethylene synthesis key gene mutants NKea-1 , the application includes improving the alkali resistance of plants.

[0013] Furthermore, the plant is selected from the leguminous plant family.

[0014] Furthermore, the legume is selected from soybeans.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: A soybean ethylene synthesis key gene mutant provided by the present invention NKea-1 , its preparation method and application, the key gene is located in the 460822~463208 base interval of soybean chromosome DNA Gm01, the mutant NKea-1 The nucleotide sequence of the mutant gene is shown in SEQ ID NO.2, and the mutant NKea-1 The amino acid sequence of the protein encoded by the coding region is shown in SEQ ID NO. 4. The present invention uses CRISPR / Cas9 gene editing technology to obtain a key gene mutant for soybean ethylene synthesis NKea-1 The mutant has greatly improved resistance to alkali stress and can be used as a parent material to cultivate new high-quality soybean varieties that are alkali-resistant or have other excellent traits through hybridization or molecular breeding.

[0016] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Picture 1 This is a schematic diagram of the structure of the key gene for ethylene synthesis and the structure of its mutants provided in Example 1 of the present invention.

[0018] Picture 2 Wm82 and mutants before and after NaHCO3 treatment provided in Detection Example 1 of the present invention NKea-1 Comparison of two soybean plants.

[0019] Picture 3 Wm82 and mutants before and after NaHCO3 treatment provided in Detection Example 1 of the present invention NKea-1 Statistical graph of the survival results of two soybean seeds.

[0020] Picture 4 Wm82 and mutants before and after NaHCO3 treatment provided in Detection Example 1 of the present invention NKea-1 Statistical graph of fresh weight results for two soybean plants. DETAILED DESCRIPTION

[0021] To make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0022] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0023] The relevant nucleotide and amino acid sequences are shown below: The key gene for soybean ethylene synthesis (nucleotides 460822 to 463208 of soybean chromosome Gm01 DNA) has a nucleotide sequence of SEQ ID NO. 1, as shown below: mutant NKea-1 The nucleotide sequence of the mutant gene is shown in SEQ ID NO. 2 as follows: mutant NKea-1 The nucleotide sequence of the cDNA is shown in SEQ ID NO. 3: mutant NKea-1 The amino acid sequence of the protein encoded by the coding region of the mutant gene is SEQ ID NO.4, as shown below: MGIEMEQPCVELSKVAVSETHGEDSPYFAGWKAYDENPYAELTNPSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKTFRTAMASFMEQVR GGRAKFDPQRVVLTAGATAANELLTFILANPGDALLVPTPYYPGFDRDLRWRTGVNIVPIHCDSSNNFQITPEALEAAYKDAEAMNSKVRGVLITNPSKPIRCNDSTFGS; The nucleotide sequence of primer DT1-BsF is SEQ ID NO.5, as shown below: ATATATGGTCTCGATTGTACACCTAATGGGTTTGAAGTT; The nucleotide sequence of primer DT1-F0 is SEQ ID NO.6, as shown below: TGTACACCTAATGGGTTTGAAGTTTTAGAGCTAGAAATAGC; The nucleotide sequence of primer DT2-R0 is SEQ ID NO.7, as shown below: AACAGGAGATGCTCTACTTGTTCAATCTCTTAGTCGACTCTAC; The nucleotide sequence of primer DT2-BsR is SEQ ID NO.8, as shown below: ATTATTGGTCTCGAAACAGGAGATGCTCTACTTGTTCAA; The nucleotide sequence of primer U6-26p-F is SEQ ID NO.9, as shown below: TGTCCCAGGATTAGAATGATTAGGC; The nucleotide sequence of primer U6-29p-R is SEQ ID NO.10, as shown below: AGCCCTCTTCTTTCGATCCATCAAC; The nucleotide sequence of primer Gm-NKea-1-F is SEQ ID NO.11, as shown below: CGCGCGATAAGTTATACTAAGAAC; The nucleotide sequence of primer Gm-NKea-1-R is SEQ ID NO.12, as shown below: AAATGGTGCCAACTCTGAAACC.

[0024] Example 1 Construction of soybean ethylene synthesis key gene mutants NKea-1 .

[0025] 1. Construct a gene editing vector.

[0026] Using CRISPR-PLANT to identify mutants of key genes for soybean ethylene biosynthesis NKea-1 sgRNAs were designed for the key genes involved in ethylene biosynthesis. Using the pCBC-DT1T2 plasmid as a template, two rounds of PCR were performed with primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR to synthesize the fragment sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2-(sgRNA-Sc). The process is as follows: The pCBC-DT1T2 plasmid was selected as the template. pCBC-DT1T2 is a vector designed for plant CRISPR / Cas system. Bsa I restriction enzyme cutting site and sgRNA scaffold (sgRNA-Sc) sequence, which can be used to construct multi-sgRNA expression cassettes; DT1-BsF and DT2-BsR were selected as outer primers to amplify the sequences of target 1 (sgRNA1) and target 2 (sgRNA2); DT1-F0 and DT2-R0 were selected as inner primers to amplify the core region of the sgRNA sequence.

[0027] The first round of PCR amplification process is as follows: The sgRNA1 sequence was amplified using DT1-BsF as the forward primer and DT1-F0 as the reverse primer to obtain U6-26p-sgRNA1-sgRNA-Sc-U6-26t. After the fragment was digested with BsaI, a sticky end was generated at the 5' end, which facilitated subsequent ligation. The sgRNA2 sequence was amplified using DT2-R0 as the forward primer and DT2-BsR (reverse) primer to obtain U6-29p-sgRNA2-sgRNA-Sc-U6-29t. This sequence was also digested with BsaI to obtain sticky ends.

[0028] The above-obtained U6-26p-sgRNA1-sgRNA-Sc-U6-26t and U6-29p-sgRNA2-sgRNA-Sc-U6-29t were mixed and a second round of PCR amplification was performed using primers DT1-BsF and DT2-BsR. The U6-26t (terminator) of sgRNA1 and the U6-29p (promoter) of sgRNA2 were seamlessly spliced ​​by sequence overlap, and the second-round PCR product sgRNA1-sgRNA-Sc-U6-26t-U6-29p-sgRNA2-sgRNA-Sc was obtained.

[0029] The second-round PCR product was subjected to a Golden Gate reaction using the restriction endonuclease Bsa I and T4 ligase provided by NEW ENGLAND Biolabs to obtain a gene editing vector. The reaction conditions were: 37°C for 5 h, 50°C for 5 min, and 80°C for 10 min.

[0030] The gene editing vector prepared above (5 μL) was transformed into competent Escherichia coli DH5α using the heat shock method, spread on LB solid plates containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Single colonies were picked and identified by PCR using primers U6-26p-F and U6-29p-R. Positive clones were shaken out, and the plasmid was extracted using the plasmid extraction kit from Quanshijin Company. The plasmid was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification to obtain the target gene editing vector.

[0031] 2. Screening and identification of soybean mutants using gene editing vectors.

[0032] The constructed gene editing vector was transformed into EHA105 Agrobacterium, and the cotyledonary node method was used to stably transform the plants. The plants were then screened for glufosinate-ammonium (Basta) resistance, and finally T0 generation resistant regenerated plants were obtained.

[0033] The harvested T0 generation seeds were disinfected with sodium hypochlorite and sown in a plastic cup filled with nutrient soil and vermiculite in a 1:1 volume ratio. When the soybean seeds germinated and the primary leaves unfolded, DNA of the T1 generation plants was extracted for PCR identification. The specific process was as follows: genomic DNA of the T1 generation transgenic plants was extracted and PCR amplified using primers Gm-NKea-1-F and Gm-NKea-1-R to obtain the mutant. NKea-1 PCR fragment of the gene target; The composition of the PCR amplification system is shown in the following table:

[0034] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s; final extension at 72°C for 10 min; and storage at 4°C.

[0035] After sequencing the amplified products, plants with mutations identified by sequencing are harvested after completing their lifecycles. The harvested seeds are sown again to produce the transgenic T2 generation. All T2 transgenic lines are subjected to PCR identification using the same method as above, and plants with homozygous mutations as sequenced are retained. The harvested seeds are sown again to produce the transgenic T3 generation. All T3 transgenic lines are subjected to PCR identification using the same method as above, and plants with homozygous mutations as sequenced are retained.

[0036] like Picture 1 The figure shows the structure of the key gene for ethylene synthesis located in the 460822~463208 base interval of soybean chromosome Gm01 and the schematic diagram of the structure of the mutant gene. The gene structure consists of exons and introns, with the start codon ATG and the stop codon TGA located at both ends of the gene. Under normal circumstances, the gene is spliced ​​to form a protein containing 446 amino acid residues. However, when the mutant NKea-1 There is a base (T) inserted in the gene sequence, which causes the amino acid sequence after position 209 to change. The final protein has only 220 amino acid residues. NKea-1 The gene has no adverse effect on the normal growth and development of soybean.

[0037] Test Example 1: Detection of alkaline stress phenotypes in plants.

[0038] 35 soybean reference varieties Wm82 and mutants with consistent full grains were selected NKea-1 seed ， Sow the seeds in a plastic cup filled with nutrient soil and vermiculite in a 1:1 ratio. When the soybean seeds germinate and the primary leaves unfold and the first three-leaf compound leaves just grow out, select 24 plants with the same developmental state. Wash the roots with clean water and transfer them to a 40mM NaHCO3 aqueous solution with a pH of 8.3. Control Wm82 and mutant NKea-1 The plants were treated with the same light duration and temperature for 58 hours and then recovered with Hoagland's nutrient solution. After 5 days of recovery, they were photographed, the survival rate was calculated, and the fresh weight was measured. The results are as follows: Picture 2 、 Picture 3 and Picture 4 As shown; Picture 2 Shows the changes of Wm82 and mutants before and after treatment with NaHCO3 NKea-1Comparison of two soybean plants. As can be seen from the figure, before treatment, the leaves of both plants were healthy, green, and neatly arranged; after treatment, the leaves of the Wm82 plant were obviously shrunken and curled, and some leaves turned yellow, showing obvious damage. NKea- 1 Although the leaves of the plant also changed, the degree of damage was significantly less than that of the Wm82 plant, and the leaves still maintained a certain green color and shape.

[0039] Picture 3 Shown are Wm82 and mutants before and after treatment with NaHCO3 NKea-1 The statistical results of the survival rates of the two soybean seeds show that the survival rate of Wm82 seeds is about 15%, and the mutant NKea-1 The seed survival rate was significantly higher than that of Wm82, about 70%. This result showed that under alkaline stress conditions, the mutant NKea-1 The seed survival rate was significantly higher than that of Wm82 mutants. NKea-1 Greater tolerance under alkaline stress.

[0040] Picture 4 Shown are Wm82 and mutants before and after treatment with NaHCO3 NKea-1 Statistical results of fresh weight of two soybean plants, mutant NKea-1 The fresh weight of the plant was about 42 g, and the fresh weight of the Wm82 plant was about 25 g. This result showed that the mutant NKea-1 It grows better in alkaline conditions.

[0041] The above results show that soybean mutant NKea-1 Showed obvious resistance to alkaline stress.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A mutant of a key gene for soybean ethylene synthesis NKea-1 , characterized in that, The key gene for soybean ethylene synthesis is located in the base interval 460822 to 463208 of the soybean chromosome DNA Gm01. NKea-1 The nucleotide sequence of the mutant gene is shown in SEQ ID NO.

2.

2. The soybean ethylene synthesis key gene mutant according to claim 1 NKea-1, Characterized by the fact that the mutant NKea-1 The amino acid sequence of the protein encoded by the coding region of the mutant gene is shown in SEQ ID NO.

4.

3. A mutant of a key gene for soybean ethylene synthesis NKea-1 The preparation method is characterized in that Used for preparing the soybean ethylene synthesis key gene mutant as claimed in claim 1 or 2 NKea-1, The steps include: S100, using CRISPR / Cas9 technology to construct mutants NKea-1 Gene editing vectors; S200, introduce gene editing vectors into plant cells through Agrobacterium-mediated plant transformation technology to screen mutants NKea-1 .

4. The soybean ethylene synthesis key gene mutant according to claim 3 NKea-1 The preparation method is characterized in that Step S100 includes the following steps: S110, using pCBC-DT1T2 as template, design primers DT1-BsF, DT1-F0, DT2-R0, and DT2-BsR; S120, amplify the sgRNA1 sequence using DT1-BsF as the forward primer and DT1-F0 as the reverse primer to obtain U6-26p-sgRNA1-sgRNA-Sc-U6-26t; The sgRNA2 sequence was amplified using DT2-R0 as the forward primer and DT2-BsR as the reverse primer to obtain U6-29p-sgRNA2-sgRNA-Sc-U6-29t; S130, U6-26p-sgRNA1-sgRNA-Sc-U6-26t and U6-29p-sgRNA2-sgRNA-Sc-U6-29t were evenly mixed, and amplified and synthesized using primers DT1-BsF and DT2-BsR to obtain sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2-(sgRNA-Sc); S140. Add Bsa I enzyme, T4 ligase, and pBSE401 vector to sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2-(sgRNA-Sc) to perform an assembly reaction to obtain a mutant. NKea-1 gene editing vectors.

5. The soybean ethylene synthesis key gene mutant according to claim 4 NKea-1 The preparation method is characterized in that In step S110, the nucleotide sequence of primer DT1-BsF is shown in SEQ ID NO.5, the nucleotide sequence of primer DT1-F0 is shown in SEQ ID NO.6, the nucleotide sequence of primer DT2-R0 is shown in SEQ ID NO.7, and the nucleotide sequence of primer DT2-BsR is shown in SEQ ID NO.

8.

6. A mutant of a key gene for soybean ethylene synthesis NKea-1 The application is characterized in that The soybean ethylene synthesis key gene mutant according to claim 1 or 2 NKea-1 , the application includes improving the alkali resistance of plants.

7. The soybean ethylene synthesis key gene mutant according to claim 6 NKea-1 The application is characterized in that The plant is selected from the family Leguminosae.

8. The soybean ethylene synthesis key gene mutant according to claim 7 NKea-1 The application is characterized in that The legume is selected from soybeans.

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