Soybean ethylene synthesis key gene mutant NKea-2 as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a soybean ethylene synthesis key gene mutant NKea-2 and a preparation method and application thereof.The soybean ethylene synthesis key gene mutant NKea-2 is obtained through the gene editing technology and comprises three mutation sequences of NKea-2-1, NKea-2-2 and NKea-2-3, NKea-2-1 is located on the Gm01 chromosome, NKea-2-2 is located on the Gm01 chromosome, NKea-2-3 is located on the Gm01 chromosome, and NKea-2-3 is located on the Gm01 chromosome. A T basic group is inserted into the nucleotide sequence of the NKea-2-2 gene, the NKea-2-2 gene is positioned on a Gm07 chromosome, an A basic group is inserted into the nucleotide sequence of the NKea-2-2 gene, the NKea-2-3 gene is positioned on a Gm08 chromosome, and a G basic group is deleted from the nucleotide sequence of the NKea-2-3 gene. The alkali stress resistance of the mutant is greatly improved, and the mutant can be used as a parent material to be applied to the cultivation of a new variety of alkali-resistant stress-resistant high-quality soybean by means of hybridization or molecular breeding and the like.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, in particular to a mutant of a key gene for soybean ethylene synthesis NKea-2 , its preparation method and application. Background Art

[0002] Soil salinization has become a major bottleneck restricting the sustainable development of global agriculture. + 、Cl - Multiple complex mechanisms, including imbalance, oxidative stress, and high pH, ​​severely inhibit normal plant growth and development. In saline-alkali environments, ion homeostasis within plant cells is disrupted, reactive oxygen species accumulate, causing oxidative damage. High pH disrupts the intracellular acid-base balance, ultimately leading to reduced or even complete crop failure, significantly limiting agricultural production efficiency and the effective use of land resources.

[0003] As an important economic crop used for both grain and oil, soybeans are not only a vital source of high-quality protein and oil for humans, but also a fundamental and strategic resource crucial to national economy and livelihoods, as well as food security. With my country's growing demand for soybeans, identifying soybean salt- and alkali-tolerant genes and exploring the functions and mechanisms of these genes are crucial for developing salt- and alkali-tolerant transgenic soybeans using genetic engineering techniques and alleviating the supply-demand imbalance in my country's soybean industry. Furthermore, these research findings can provide theoretical and technical insights for the development of other salt- and alkali-tolerant crops, significantly promoting the efficient utilization of saline-alkali land resources and sustainable agricultural development. Ethylene, one of the oldest gaseous hormones in plants, has a simple chemical structure and plays a key regulatory role in physiological processes such as plant growth and development and defense responses. Previous studies in the model plant Arabidopsis thaliana have shown that various abiotic stresses can significantly affect the expression of different genes of key enzymes in ethylene synthesis. However, to date, no relevant research has been reported on the response mechanism of soybean ethylene synthesis-related genes to saline-alkali stress, and how to use soybean ethylene synthesis-related genes to carry out salt-alkali tolerance breeding is also a blank. Therefore, in-depth research on the function of soybean ethylene synthesis-related genes under saline-alkali stress is of great significance for breeding new salt-alkali-tolerant soybean varieties and breaking the restrictions on saline-alkali soil cultivation. Summary of the Invention

[0004] 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-2 The second object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-2 The third object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-2 application.

[0005] 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-2 , the mutant NKea-2 include NKea-2-1, NKea-2-2 and NKea-2-3 Three mutation sequences; in, NKea-2-1 Located in the DNA base interval 460822 to 463208 on chromosome Gm01, the nucleotide sequence of which is shown in SEQ ID NO.1; NKea-2-2 Located in the DNA base interval 15273601 to 15275841 on chromosome Gm07, the nucleotide sequence of which is shown in SEQ ID NO.2; NKea-2-3 Located in the DNA base interval 2422317 to 2424407 on chromosome Gm08, its nucleotide sequence is shown in SEQ ID NO.3 Furthermore, NKea-2-1 The middle coding region encodes mutant protein I, whose amino acid sequence is shown in SEQ ID NO.4; NKea-2-2 The middle coding region encodes mutant protein II, whose amino acid sequence is shown in SEQ ID NO.5; NKea-2-3 The middle coding region encodes mutant protein III, and its amino acid sequence is shown in SEQ ID NO.6.

[0006] 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-2 A preparation method for preparing any of the above soybean ethylene synthesis key gene mutants NKea-2, The steps include: S100, using CRISPR / Cas9 technology to construct mutants NKea-2 Gene editing vectors; S200, introduce gene editing vectors into plant cells through Agrobacterium-mediated plant transformation technology to screen mutants NKea-2 .

[0007] Furthermore, step S100 includes the following steps: S110, using pCBC-DT1T2 as a template and primers EA-F, EA-F0, EA-R0, and EA-R, two rounds of PCR amplification were performed to obtain the PCR product sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2; S120, the PCR product was reacted with the pBSE401 vector by Golden Gate reaction to obtain the mutant NKea-2 gene editing vectors.

[0008] Furthermore, in step S110, the nucleotide sequence of primer EA-F is shown as SEQ ID NO.7, the nucleotide sequence of primer EA-F0 is shown as SEQ ID NO.8, the nucleotide sequence of primer EA-R0 is shown as SEQ ID NO.9, and the nucleotide sequence of primer EA-R is shown as SEQ ID NO.10.

[0009] Furthermore, in step S200, the mutants are screened using molecular screening methods. NKea-2 to filter.

[0010] Further, during the molecular screening process, the molecular identification primers were selected from 3900-F, 3900-R, 8000-F, 8000-R, 0100-F, and 0100-R; The nucleotide sequence of 3900-F is shown in SEQ ID NO.11; The nucleotide sequence of 3900-R is shown in SEQ ID NO. 12; The nucleotide sequence of 8000-F is shown in SEQ ID NO. 13; The nucleotide sequence of 8000-R is shown in SEQ ID NO. 14; The nucleotide sequence of 0100-F is shown in SEQ ID NO. 15; The nucleotide sequence of 0100-R is shown in SEQ ID NO.16.

[0011] 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-2 Application, such as any of the above soybean ethylene synthesis key gene mutants NKea-2 , the application includes improving the alkali resistance of plants.

[0012] Furthermore, the plant is selected from the leguminous plant family.

[0013] Furthermore, the legume is selected from soybeans.

[0014] 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-2 , its preparation method and application, mutant NKea-2 include NKea-2-1, NKea-2-2 and NKea-2-3 Three mutation sequences; NKea-2-1 Located in the 460822-463208 base range of the DNA on chromosome Gm01, a T base is inserted after the 462036 base of the DNA on chromosome Gm01, causing the translation product of the key gene for ethylene synthesis encoded within the nucleotide range of the chromosome to mutate after the 208th amino acid, and the translation is terminated prematurely at the 220th amino acid; NKea-2-2 Located in the 15273601 to 15275841 base range of the DNA on chromosome Gm07, an A base is inserted after the 15274674 base of the DNA on chromosome Gm07, causing the translation product of the key gene protein for ethylene synthesis encoded by the nucleotide inversion interval in the chromosome DNA fragment to mutate after amino acid 210, and the translation is terminated prematurely at amino acid 220; NKea-2-3 Located in the base range of 2422317 to 2424407 of the DNA on chromosome Gm08, a G base is deleted at position 2423098 of the DNA on chromosome Gm08, causing the translation product of the key gene for ethylene synthesis encoded within the nucleotide range of this chromosome to mutate after amino acid 144 and terminate prematurely at amino acid 160.

[0015] The present invention uses CRISPR / Cas9 gene editing technology to obtain soybean ethylene synthesis key gene mutants NKea-2 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 It is the key gene mutant of ethylene synthesis provided in Example 1 of the present invention NKea-2 Schematic diagram of the gene editing sites.

[0018] Picture 2 Wm82 and mutants before and after NaHCO3 treatment provided in Detection Example 1 of the present invention NKea-2 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-2 Statistical graph of the survival results of two soybean seeds. DETAILED DESCRIPTION

[0020] 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.

[0021] 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.

[0022] The relevant nucleotide and amino acid sequences are shown below: NKea-2-1 (located in the 460822-463208 base interval of the DNA on soybean chromosome Gm01), the nucleotide sequence of which is shown in the following SEQ ID NO.1: NKea-2-2 (located in the DNA base interval 15273601 to 15275841 on soybean chromosome Gm07), the nucleotide sequence of which is shown in SEQ ID NO. 2 below: NKea-2-3 (located in the DNA base interval 2422317 to 2424407 on soybean chromosome Gm08), the nucleotide sequence of which is shown in SEQ ID NO.3 below: The amino acid sequence of the mutant protein I on chromosome Gm01 is shown in SEQ ID NO. 4 below: MGIEMEQPCVELSKVAVSETHGEDSPYFAGWKAYDENPYAELTNPSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKTFRTAMASFMEQVR GGRAKFDPQRVVLTAGATAANELLTFILANPGDALLVPTPYYPGFDRDLRWRTGVNIVPIHCDSSNNFQITPEALEAAYKDAEAMNSKVRGVLITNPSKPIRCNDSTFGS; The amino acid sequence of the mutant protein II on chromosome Gm07 is shown in the following SEQ ID NO.5: MGIEMEQPCVELSKVAVSETHGEDSPYFAGWKAYDENPYDELTNPSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKTFRTAMASFMEQVR GGRAKFDPQRLVLTAGATAANELLTFILANPGDALLVPTPYYPGFDRDLRWRTGVNIVPIHCDSSNNFQITPEALEAAYKDAEAMNSKVRGVLITNPSNPIRCNNSTFGS; The amino acid sequence of the mutant protein III on chromosome Gm08 is shown in SEQ ID NO.6 below: MGIKIEQEQPCVELSRVAVSETHGEDSPYFAGWKAYDENPYDELTNSSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKSFRTAMASFMEQIRGGRAKFDPDRVVLTAGATAANELLTFILANPGMLYLFQRLTIQDLIEI.

[0023] The nucleotide sequence of primer EA-F is SEQ ID NO.7, as shown below: ATATATGGTCTCGATTGTACACCTAATGGGTTTGAAGTT; The nucleotide sequence of primer EA-F0 is SEQ ID NO.8, as shown below: TGTACACCTAATGGGTTTGAAGTTTTAGAGCTAGAAATAGC; The nucleotide sequence of primer EA-R0 is SEQ ID NO.9, as shown below: AACAGGAGATGCTCTACTTGTTCAATCTCTTAGTCGACTCTAC; The nucleotide sequence of primer EA-R is SEQ ID NO.10, as shown below: ATTATTGGTCTCGAAACAGGAGATGCTCTACTTGTTCAA; The nucleotide sequence of primer 3900-F is SEQ ID NO.11, as shown below: CGCGCGATAAGTTATACTAAGAAC; The nucleotide sequence of primer 3900-R is SEQ ID NO.12, as shown below: AAATGGTGCCAACTCTGAAACC; The nucleotide sequence of primer 8000-F is SEQ ID NO.13, as shown below: AGTGTGAGAGAGAAAGTGTCATC; The nucleotide sequence of primer 8000-R is SEQ ID NO.14, as shown below: AGTTCTCGGTGAACTTCTTATCC; The nucleotide sequence of primer 0100-F is SEQ ID NO.15, as shown below: TCCTCTCGGAAAGAGTTGCAA; The nucleotide sequence of primer 0100-R is SEQ ID NO.16, as shown below: GTGATTTGGAAGTTGTTTGAGC; The nucleotide sequence of primer U6-26p-F is SEQ ID NO.17, as shown below: TGTCCCAGGATTAGAATGATTAGGC; The nucleotide sequence of primer U6-29p-R is SEQ ID NO.18, as shown below: AGCCCTCTTCTTTCGATCCATCAAC.

[0024] Example 1 Construction of soybean ethylene synthesis key gene mutants NKea-2 .

[0025] 1. Construct a gene editing vector.

[0026] The online tool CRISPR-PLANT was used to perform a genomic sequencing of the soybean chromosome Gm01 DNA at bases 460822 to 463208. 、 sgRNAs were designed for three key ethylene biosynthesis genes with high homology in the base interval 15273601 to 15275841 of chromosome DNA Gm07 and the base interval 2422317 to 2424407 of chromosome DNA Gm08, and the above genes were knocked out and edited by two sgRNAs. Using the pCBC-DT1T2 plasmid as a template, two rounds of PCR were performed with primers EA-F (SEQ ID NO.7), EA-F0 (SEQ ID NO.8), EA-R0 (SEQ ID NO.9), and EA-R (SEQ ID NO.10) to synthesize the fragment sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2, and the fragments were added at both ends. Bsa I restriction enzyme cutting site.

[0027] The PCR reaction system is shown in the following table: The PCR reaction conditions are as follows: The PCR fragment was 626 bp in length. After cleaning the PCR product, the restriction enzymes from NEW ENGLAND Biolabs were used to Bsa The Golden Gate reaction was performed using T4 DNA Ligase (HC, NEB) and I enzyme. The reaction system is shown in the following table: The reaction conditions were as follows: incubation at 37°C for 5 h, incubation at 50°C for 5 min, and inactivation at 80°C for 10 min.

[0028] 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 (SEQ ID NO. 17) and U6-29p-R (SEQ ID NO. 18). 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.

[0029] 2. Screening and identification of gene-edited soybean mutants.

[0030] 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.

[0031] Genomic DNA was extracted from the offspring of the resistant plants, and molecular identification was performed through PCR and sequencing to screen for mutant materials with different forms of editing in the target gene. A single mutant with a T base inserted at position 462036 of the Gm01 chromosome DNA and a double mutant with an A base inserted at position 15274674 of the Gm07 chromosome DNA and a G base deleted at position 2423098 of the Gm08 chromosome DNA were screened. After screening the above homozygous mutant materials, hybrid offspring were obtained through genetic hybridization technology, and identified through PCR and sequencing to screen for homozygous mutants. NKea-2 .

[0032] mutant NKea-2 1 , and the editing situation is as follows: a T base is inserted at position 462036 of the chromosome DNA of Gm01, resulting in a mutation in the translation product of the key gene for ethylene synthesis encoded within the base range of 460822 to 463208 of the chromosome DNA after amino acid 208, and premature termination of translation at amino acid 220; an A base is inserted at position 15274674 of the chromosome DNA of Gm07, resulting in a mutation in the translation product of the key gene protein for ethylene synthesis encoded within the reverse complementary range of bases 15273601 to 15275841 in the chromosome DNA fragment after amino acid 210, and premature termination of translation at amino acid 220; a G base is deleted at position 2423098 of the chromosome DNA of Gm08, resulting in a mutation in the translation product of the key gene for ethylene synthesis in the base range of 2422317 to 2424407 after amino acid 144, and premature termination of translation at amino acid 160.

[0033] mutant NKea-2 The molecular identification primers were 3900-F (SEQ ID NO.11), 3900-R (SEQ ID NO.12), 8000-F (SEQ ID NO.13), 8000-R (SEQ ID NO.14), 0100-F (SEQ ID NO.15), and 0100-R (SEQ ID NO.16); Test Example 1: Detection of alkaline stress phenotypes in plants.

[0034] 35 soybean reference varieties Wm82 and mutants with consistent full grains were selected NKea-2 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-2 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 weighed. The results are as follows: Picture 2 and Picture 3 shown.

[0035] Picture 2 Shows the changes of Wm82 and mutants before and after treatment with NaHCO3 NKea-2 Comparison 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- 2 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.

[0036] Picture 3 Shown are Wm82 and mutants before and after treatment with NaHCO3 NKea-2 The statistical results of the survival rates of the two soybean seeds show that the survival rate of Wm82 seeds is about 19%, and the mutant NKea-2 The seed survival rate was significantly higher than that of Wm82, about 90%. This result showed that under alkaline stress conditions, the mutant NKea-2 The seed survival rate was significantly higher than that of Wm82 mutants. NKea-2 It has a stronger tolerance to alkaline stress.

[0037] The above results show that soybean mutant NKea-2 Showed obvious resistance to alkaline stress.

[0038] 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-2 , characterized in that, The mutant NKea-2 include NKea-2-1, NKea-2-2 and NKea-2-3 Three mutation sequences; in, NKea-2-1 Located in the DNA base interval 460822 to 463208 on chromosome Gm01, the nucleotide sequence of which is shown in SEQ ID NO.1; NKea-2-2 Located in the DNA base interval 15273601 to 15275841 on chromosome Gm07, the nucleotide sequence of which is shown in SEQ ID NO.2; NKea-2-3 It is located in the base interval 2422317 to 2424407 of the DNA on chromosome Gm08, and its nucleotide sequence is shown in SEQ ID NO.

3.

2. The soybean ethylene synthesis key gene mutant according to claim 1 NKea-2, It is characterized in that NKea-2-1 The middle coding region encodes mutant protein I, whose amino acid sequence is shown in SEQ ID NO.4; NKea-2-2 The middle coding region encodes mutant protein II, whose amino acid sequence is shown in SEQ ID NO.5; NKea-2-3 The middle coding region encodes mutant protein III, and its amino acid sequence is shown in SEQ ID NO.

6.

3. A mutant of a key gene for soybean ethylene synthesis NKea-2 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-2, The steps include: S100, using CRISPR / Cas9 technology to construct mutants NKea-2 Gene editing vectors; S200, introduce gene editing vectors into plant cells through Agrobacterium-mediated plant transformation technology to screen mutants NKea-2 .

4. The soybean ethylene synthesis key gene mutant according to claim 3 NKea-2 The preparation method is characterized in that Step S100 includes the following steps: S110, using pCBC-DT1T2 as a template and primers EA-F, EA-F0, EA-R0, and EA-R, two rounds of PCR amplification were performed to obtain the PCR product sgRNA1-(sgRNA-Sc)-(U6-26t)-(U6-29p)-sgRNA2; S120, the PCR product was reacted with the pBSE401 vector by Golden Gate reaction to obtain the mutant NKea-2 gene editing vectors.

5. The soybean ethylene synthesis key gene mutant according to claim 4 NKea-2 The preparation method is characterized in that In step S110, the nucleotide sequence of primer EA-F is shown in SEQ ID NO.7, the nucleotide sequence of primer EA-F0 is shown in SEQ ID NO.8, the nucleotide sequence of primer EA-R0 is shown in SEQ ID NO.9, and the nucleotide sequence of primer EA-R is shown in SEQ ID NO.

10.

6. The soybean ethylene synthesis key gene mutant according to claim 3 NKea-2 The preparation method is characterized in that In step S200, the mutant is screened using a molecular screening method. NKea-2 to filter.

7. The soybean ethylene synthesis key gene mutant according to claim 6 NKea-2 The preparation method is characterized in that During the molecular screening process, the molecular identification primers were selected from 3900-F, 3900-R, 8000-F, 8000-R, 0100-F, and 0100-R; The nucleotide sequence of 3900-F is shown in SEQ ID NO.11; The nucleotide sequence of 3900-R is shown in SEQ ID NO. 12; The nucleotide sequence of 8000-F is shown in SEQ ID NO. 13; The nucleotide sequence of 8000-R is shown in SEQ ID NO. 14; The nucleotide sequence of 0100-F is shown in SEQ ID NO. 15; The nucleotide sequence of 0100-R is shown in SEQ ID NO.

16.

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

9. The soybean ethylene synthesis key gene mutant according to claim 8 NKea-2 The application is characterized in that The plant is selected from the family Leguminosae.

10. The soybean ethylene synthesis key gene mutant according to claim 9 NKea-2 The application is characterized in that The legume is selected from soybeans.

Images