Soybean ethylene synthesis key gene mutant NKea-3 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-3 and a preparation method and application thereof, the mutant NKea-3 comprises four mutation sites of a genome mutation site I, a mutation site II, a mutation site III and a mutation site IV; a basic group A is inserted into the mutation site I; a base G and a base A are deleted at the mutation site II; a basic group A is inserted into the mutation site III; and a base G is deleted at the mutation site IV. The method comprises the following steps: editing four ethylene synthesis key genes in soybean by using a CRISPR / Cas9 gene editing technology to obtain a mutant NKea-3; a stress resistance detection experiment result of the mutant NKea-3 shows that the resistance of the mutant to both alkali stress and drought stress is remarkably enhanced. Therefore, the gene is expected to be used as a parent material for improving the alkali stress and / or drought stress resistance of crops through 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-3 and its preparation method and application. Background Art

[0002] Abiotic stresses are adverse conditions in a plant's growth environment caused by non-biological factors. These include extreme temperatures, drought, salinity, flooding, light intensity, soil pH, and heavy metal pollution. These conditions severely restrict plant growth and crop yields. Among these stressors, alkaline stress and drought stress are two widespread and significant abiotic stresses. They not only directly affect plant growth and development but also pose a serious threat to global ecosystem stability and the sustainability of agricultural production. Globally, salinized land is widely distributed. The difference between alkaline stress and salt stress is that in addition to ion toxicity and osmotic stress, high pH stress is also superimposed, so its comprehensive harm is significantly higher than that of single salt stress or pH stress. High pH will destroy the permeability of plant cell membranes and inhibit the root system's absorption of Ca 2+ Mg 2+ The absorption of essential mineral elements such as ions will lead to ion imbalance in the plant body. At the same time, the high pH environment will reduce the oxygen solubility in the rhizosphere microenvironment, further inhibiting the respiratory metabolism of the root system. Alkali stress will reduce the soil osmotic potential, causing water loss in plant cells, resulting in osmotic stress, and high concentrations of Na + It will also interfere with K + The absorption of ions disrupts the ion balance in plants. Furthermore, alkaline stress can cause changes in plant leaf cell structure, a decrease in photosynthetic rate, and a reduction in chlorophyll content, hindering photosynthesis. It can also promote the accumulation of reactive oxygen species (ROS) in plants, triggering cell membrane lipid peroxidation, increasing membrane permeability, and causing serious damage to plant cells.

[0003] Drought stress has multi-dimensional characteristics in its damage to plants. First, water deficiency directly causes cell osmotic stress, leading to stomatal closure, inhibiting photosynthesis and aggravating ROS accumulation, causing oxidative damage; second, the decline in root water absorption capacity will disrupt the body's ion balance (such as K + / Na +These effects not only restrict plant growth but also pose a significant challenge to the sustainability of agricultural production. In some arid regions, water scarcity limits crop growth and the sustainable use of farmland. When these two stresses occur in combination, the suppression of crop growth and yield is far greater than that of either stress alone.

[0004] As one of the oldest gaseous hormones in plants, ethylene has a simple chemical structure and plays a key regulatory role in physiological processes such as plant growth and development and defense response. 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, the current research on the function of soybean ethylene synthesis-related genes in alkali-tolerant and drought-tolerant breeding is still a blank. Exploring key genes for salt-alkali tolerance and drought resistance and developing molecular breeding strategies to cultivate new multi-resistant varieties are important ways to alleviate the threat of abiotic stress to crops. Therefore, in-depth research on the function of soybean ethylene synthesis-related genes under abiotic stress is of great significance for breeding new alkali-tolerant and drought-tolerant plant varieties and breaking through the limitations of planting in saline-alkali and arid lands. Summary of the Invention

[0005] 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-3 The second object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-3 The third object of the present invention is to provide a soybean ethylene synthesis key gene mutant NKea-3 application.

[0006] 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-3 , the mutant NKea-3 Including four mutation sites: genomic mutation site I, mutation site II, mutation site III and mutation site IV; Among them, mutation site Ⅰ is located at the last position of DNA 462039 of chromosome Gm01, and a base A is inserted at mutation site Ⅰ; Mutation site II is located at positions 42454464 and 42454465 of chromosome DNA of Gm05, where one base G and one base A are deleted; Mutation site III is located after position 15274674 of the DNA of chromosome Gm07, and a base A is inserted at mutation site III; Mutation site IV is located at position 2423098 of chromosome DNA Gm08, and a base G is missing at mutation site IV.

[0007] Furthermore, the mutation site I is located above the mutation sequence I, and the nucleotide sequence of the mutation sequence I is shown in SEQ ID NO.1; The mutation site II is located above the mutation sequence II, and the nucleotide sequence of the mutation sequence II is shown in SEQ ID NO.2; The mutation site III is located above the mutation sequence III, and the nucleotide sequence of the mutation sequence III is shown in SEQ ID NO. 3; The mutation site IV is located above the mutation sequence IV, and the nucleotide sequence of the mutation sequence IV is shown in SEQ ID NO.4.

[0008] Furthermore, the mutant sequence I encodes mutant protein I, whose amino acid sequence is shown in SEQ ID NO.5; the mutant sequence II encodes mutant protein II, whose amino acid sequence is shown in SEQ ID NO.6; the mutant sequence III encodes mutant protein III, whose amino acid sequence is shown in SEQ ID NO.7; and the mutant sequence IV encodes mutant protein IV, whose amino acid sequence is shown in SEQ ID NO.8.

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

[0010] Furthermore, step S100 includes the following steps: S110, using pCBC-DT1T2 as a template, two rounds of PCR amplification were performed with primers 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-3 gene editing vectors.

[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-3 Application, such as any of the above soybean ethylene synthesis key gene mutants NKea-3 The application includes improving the alkali stress resistance and / or drought stress resistance of crops.

[0012] Furthermore, improving the alkali stress resistance and / or drought stress resistance of crops includes breeding improved varieties of crops.

[0013] Furthermore, the crops are selected from one or more of leguminous crops, rice, corn, wheat, tomato, pineapple, cucumber, potato, sugarcane and cotton.

[0014] Furthermore, the legume crops are selected from one or more of soybeans, broad beans, peas, mung beans, adzuki beans, peas, pigeon peas and chickpeas.

[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-3 , its preparation method and application, mutant NKea-3 It includes four mutation sites: genomic mutation site I, mutation site II, mutation site III and mutation site IV; mutation site I is located at the position after 462039 of chromosome DNA of Gm01, and mutation site I is an insertion of a base A; mutation site II is located at positions 42454464 and 42454465 of chromosome DNA of Gm05, and a base G and a base A are deleted at mutation site II; mutation site III is located at the position after 15274674 of chromosome DNA of Gm07, and a base A is inserted at mutation site III; mutation site IV is located at the position after 2423098 of chromosome DNA of Gm08, and a base G is deleted at mutation site IV.

[0016] The results of the alkaline stress resistance and drought stress resistance experiments showed that the mutants prepared by knocking out four key ethylene synthesis genes in soybean using CRISPR / Cas9 gene editing technology NKea-3 The mutant has significant resistance to both alkali and drought stress. Therefore, it is expected to be used as a parent material to improve the alkali and / or drought stress resistance of crops through hybridization or molecular breeding.

[0017] 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

[0018] Picture 1It is the key gene mutant of ethylene synthesis provided in Example 1 of the present invention NKea-3 Schematic diagram of the gene editing sites.

[0019] Picture 2 The control Williams82 (Wm82) and mutants were treated with NaHCO3 before and after in the hydroponic experiment provided in Test Example 1 of the present invention. NKea-3 Comparison of two soybean plants.

[0020] Picture 3 The results are as follows: Wm82 and mutants before and after NaHCO3 treatment in the saline-alkali soil experiment provided in Test Example 1 of the present invention NKea-3 Comparison of two soybean plants.

[0021] Picture 4 Wm82 and mutants before and after drought treatment provided in Test Example 2 of the present invention NKea-3 Comparison of two soybean plants. DETAILED DESCRIPTION

[0022] 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 specific embodiments. 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.

[0023] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used are all commercially available unless otherwise specified.

[0024] The relevant nucleotide and amino acid sequences are shown below: Mutant sequence I (located in the base range 460822 to 463208 of the DNA on soybean chromosome Gm01) has a nucleotide sequence as shown in SEQ ID NO. 1 below: Mutant sequence II (located in the base range 460822 to 463208 of the DNA on soybean chromosome Gm05) has a nucleotide sequence as shown in SEQ ID NO. 2 below: Mutant sequence III (located in the base interval 15273601 to 15275841 of the DNA on soybean chromosome Gm07) has a nucleotide sequence as shown in SEQ ID NO. 3 below: Mutant sequence IV (located in the base interval 2422317 to 2424407 of the DNA on soybean chromosome Gm08) has a nucleotide sequence as shown in SEQ ID NO. 4 below: The amino acid sequence of the mutant protein I on chromosome Gm01 is shown in SEQ ID NO. 5 below: MGIEMEQPCVELSKVAVSETHGEDSPYFAGWKAYDENPYAELTNPSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKTFRTAMASFMEQVR GGRAKFDPQRVVLTAGATAANELLTFILANPGDALLVPTPYYPGFDRDLRWRTGVNIVPIHCDSSNNFQITPEALEAAYKDAEAMNSKVRGVLITNPSKPIRCNDSTFGS; The amino acid sequence of the mutant protein II on chromosome Gm05 is shown in SEQ ID NO.6 below: MGIKIEQEQPSVELSRIAVSETHGEHSPYFAGWKAYDENPYDELTNSSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKSFRTAMASFMEQIRGGRAKFDPDRVVLTAGATAANELLTFILANPGCSTCSNPLLSRI; The amino acid sequence of the mutant protein III on chromosome Gm07 is shown in SEQ ID NO.7 below: MGIEMEQPCVELSKVAVSETHGEDSPYFAGWKAYDENPYDELTNPSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKTFRTAMASFMEQVR GGRAKFDPQRLVLTAGATAANELLTFILANPGDALLVPTPYYPGFDRDLRWRTGVNIVPIHCDSSNNFQITPEALEAAYKDAEAMNSKVRGVLITNPSNPIRCNNSTFGS; The amino acid sequence of the mutant protein IV on chromosome Gm08 is shown in SEQ ID NO.8 below: MGIKIEQEQPCVELSRVAVSETHGEDSPYFAGWKAYDENPYDELTNSSGVIQMGLAENQVSFDLLEKYLEEHSEASTWGKGAPGFRENALFQDYHGLKSFRTAMASFMEQIRGGRAKFDPDRVVLTAGATAANELLTFILANPGMLYLFQRLTIQDLIEI; The nucleotide sequence of primer EA-3F is SEQ ID NO.9, as shown below: ATATATGGTCTCGATTGTACACCTAATGGGTTTGAAGTT; The nucleotide sequence of primer EA-3F0 is SEQ ID NO.10, as shown below: TGTACACCTAATGGGTTTGAAGTTTTAGAGCTAGAAATAGC; The nucleotide sequence of primer EA-3R0 is SEQ ID NO.11, as shown below: AACAGGAGATGCTCTACTTGTTCAATCTCTTAGTCGACTCTAC; The nucleotide sequence of primer EA-3R is SEQ ID NO.12, as shown below: ATTATTGGTCTCGAAACAGGAGATGCTCTACTTGTTCAA; The nucleotide sequence of primer U6-26p-F is SEQ ID NO.13, as shown below: TGTCCCAGGATTAGAATGATTAGGC; The nucleotide sequence of primer U6-29p-R is SEQ ID NO.14, as shown below: AGCCCTCTTCTTTCGATCCATCAAC; The nucleotide sequence of primer EA3-1-F is SEQ ID NO.15, as shown below: CGCGCGATAAGTTATACTAAGAAC; The nucleotide sequence of primer EA3-1-R is SEQ ID NO.16, as shown below: AAATGGTGCCAACTCTGAAACC; The nucleotide sequence of primer EA3-2-F is SEQ ID NO.17, as shown below: TGTACCCATCAGGTTTCATTTG; The nucleotide sequence of primer EA3-2-R is SEQ ID NO.18, as shown below: AGCTTGAGGAGTGATTTGGAAG; The nucleotide sequence of primer EA3-3-F is SEQ ID NO.19, as shown below: AGTGTGAGAGAGAAAGTGTCATC; The nucleotide sequence of primer EA3-3-R is SEQ ID NO.20, as shown below: AGTTCTCGGTGAACTTCTTATCC; The nucleotide sequence of primer EA3-4-F is SEQ ID NO.21, as shown below: TCCTCTCGGAAAGAGTTGCAA; The nucleotide sequence of primer EA3-4-R is SEQ ID NO.22, as shown below: GTGATTTGGAAGTTGTTTGAGC; Example 1 Construction of soybean ethylene synthesis key gene mutants NKea-3 .

[0025] 1. Construct the target gene editing vector.

[0026] The CRISPR-PLANT online tool was used to design sgRNAs for key genes in ethylene synthesis. Two sgRNAs were designed to knock out key genes in ethylene synthesis. Using the pCBC-DT1T2 plasmid as a template, two rounds of PCR were performed with primers EA3F (as shown in SEQ ID NO.9), EA3F0 (as shown in SEQ ID NO.10), EA3R0 (as shown in SEQ ID NO.11), and EA3R (as shown in SEQ ID NO.12) 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 length of the PCR fragment was 626 bp. After the PCR product was purified, the restriction endonuclease 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. 13) and U6-29p-R (SEQ ID NO. 14). 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] To obtain T0 generation plants, the process is as follows: the constructed gene editing vector is transformed into EHA105 Agrobacterium, and the cotyledonary node method is used to stably transform the plants, followed by glufosinate-ammonium (Basta) resistance screening, to finally obtain T0 generation resistant regenerated plants.

[0031] Screening of T1 plants involved the following process: molecular characterization of T0 plants was performed, and strains demonstrating gene editing were retained. Harvested T0 seeds were disinfected with sodium hypochlorite and sown in plastic cups containing soil amended with nutrient soil and vermiculite (at a 1:1 volume ratio). DNA from T1 plants was extracted after germination and initial leaves expanded, and analyzed by PCR and sequencing. Plants demonstrating gene editing at all four target editing sites were cultured until seed harvest.

[0032] To obtain T2 generation plants, the process is as follows: the harvested seeds are sown again to obtain transgenic T2 generation plants, and the transgenic lines of the T2 generation are again identified by PCR. The mutants screened in the T2 generation are identified by PCR, and the lines in which the editor has been isolated are screened.

[0033] After PCR and sequencing identification of the editing sites of the four key genes in the T1 and T2 generations, quadruple mutants with all four genes edited were obtained. NKea-3 The homozygous lines were harvested and seeds were obtained.

[0034] mutant NKea-3The editing forms of each gene are as follows: an A is inserted after base 462039 of chromosome Gm01, which causes the translation product of the key gene for ethylene synthesis encoded in the chromosomal DNA interval of 460822-463208 to mutate after amino acid 208, and the translation is terminated prematurely at amino acid 220; a G and an A are deleted at positions 42454464 and 42454465 of chromosome DNA of Gm05, respectively, which causes the translation product of the key gene for ethylene synthesis encoded in the chromosomal DNA interval of 42453584-42455777 to mutate after amino acid 144, and to terminate the translation at amino acid 156. Amino acid translation terminates prematurely; 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 in the reverse complementary interval of bases 15273601 to 15275841 in the chromosome DNA fragment after amino acid 210, and the translation terminates prematurely 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 region 2422317 to 2424407 after amino acid 144, and terminates prematurely at amino acid 160.

[0035] The above mutants NKea-3 The molecular identification primer sequences are EA3-1-F (as shown in SEQ ID NO.15), EA3-1-R (as shown in SEQ ID NO.16), EA3-2-F (as shown in SEQ ID NO.17), EA3-2-R (as shown in SEQ ID NO.18), EA3-3-F (as shown in SEQ ID NO.19), EA3-3-R (as shown in SEQ ID NO.20), EA3-4-F (as shown in SEQ ID NO.21), and EA3-4-R (as shown in SEQ ID NO.22). The editing sites are shown in FIG. Picture 1 As shown, ** in the figure represents GA, and * in the figure represents G.

[0036] Test Example 1: Mutant NKea-3 Test of alkaline stress resistance.

[0037] 35 full and consistent control Williams82 (Wm82) and mutants were selected respectively. NKea-3 Sow the seeds of soybeans in plastic cups containing soil modified with nutrient soil and vermiculite (a 1:1 volume ratio). Once the soybean seeds germinate, their primary leaves unfold, and the first trifoliate leaves emerge, select 24 plants of identical developmental status. The two groups of 24 plants were then subjected to alkaline stress treatments in hydroponics and soil cultivation, respectively.

[0038] The process of hydroponic alkaline stress treatment is as follows: NKea-3 The roots of the plants and the Wm82 control plants were washed with clean water and then transferred to a 40 mM NaHCO3 aqueous solution with a pH of 8.3. The light duration and temperature of the wild type and mutant materials were controlled to be the same. After 58 hours of cultivation, they were recovered with Hoagland's nutrient solution and observed after 5 days of recovery. The results are as follows Picture 2 As shown; in the figure, Wm82 and mutants before treatment NKea-3 The leaves of Wm82 showed similar morphology, all showing a healthy and stretched state; after treatment, the leaves of Wm82 showed obvious withering and decay, and the growth was highly inhibited; while the mutant NKea-3 Although some leaves were affected, more healthy leaves were retained overall, and the degree of growth inhibition was relatively low; Picture 2 The results showed that under 40 mM NaHCO3 alkaline stress, the mutant NKea-3 It has stronger alkali resistance than Wm82. The mutation gives the plant an advantage in coping with alkaline environmental stress, which is beneficial for it to maintain growth under alkaline stress.

[0039] The process of soil culture alkali stress treatment is as follows: the roots of the mutant plants and the control plants in another group were washed according to the hydroponic treatment method, and then transferred to saline-alkali soil with a pH of 9.7 prepared with a 60 mM NaHCO3 + 15 mM Na2CO3 aqueous solution. Picture 3 As shown in the figure, it can be seen that the leaves of Wm82 are yellowing and withering. NKea-3 The green leaves are more, indicating that under this alkaline stress condition, the mutant NKea-3 The alkali resistance (adversity resistance) is significantly better than Wm82.

[0040] In summary, whether it is hydroponic or soil culture, the mutant NKea-3 The alkaline stress tolerance of the strains was significantly stronger than that of Wm82.

[0041] Test Example 2: Mutant NKea-3 Test of drought stress resistance.

[0042] Wm82 and mutants were selected NKea-3 The full and uniform seeds were sown in a plastic cup filled with soil modified with nutrient soil and vermiculite (the volume ratio of the two was 1:1). After the soybean seeds germinated and the primary leaves unfolded, drought stress treatment was carried out until Wm82 was almost completely wilted. At this time, the mutant NKea-3 Most of the strains showed more obvious drought resistance. At this time, after rehydration treatment, mutants were found. NKea-3The drought stress resistance of Picture 4 As shown; From the pictures before drought treatment, we can see that Wm82 and mutant NKea-3 The growth of the plants was similar; after drought stress treatment, the plants of Wm82 withered and their growth was severely inhibited, while the mutant NKea-3 Although the plants were also affected by drought, their overall condition was better than that of Wm82 plants. This result showed that the mutant NKea-3 It can retain more vitality under drought stress; after rehydration, Wm82 plants also recovered to a certain extent, but the mutant NKea-3 The plants recovered more obviously and were stronger; the results showed that the mutant NKea-3 It has stronger drought resistance and its ability to recover after drought is significantly better than Wm82; The process of drought stress treatment was as follows: the water content of the amended soil was gradually reduced to 20%–15% (maintaining water content); The rehydration process is as follows: the moisture content of the improved soil is increased to 60% to 75% (maintaining water content).

[0043] The experimental results of Test Example 1 and Test Example 2 show that the mutant NKea-3 It has no adverse effect on the normal growth and development of soybeans, and significantly improves the resistance to alkaline stress and drought stress. Therefore, the present invention provides a knockout mutant of four key genes for soybean ethylene synthesis obtained by gene editing. NKea-3 , which is expected to be used to improve soybean's resistance to alkali stress, drought stress, and combined resistance to alkali stress and drought stress.

[0044] 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-3 , characterized in that, The mutant NKea-3 Including four mutation sites: genomic mutation site I, mutation site II, mutation site III and mutation site IV; Among them, mutation site Ⅰ is located at the last position of DNA 462039 of chromosome Gm01, and a base A is inserted at mutation site Ⅰ; Mutation site II is located at positions 42454464 and 42454465 of chromosome DNA of Gm05, where one base G and one base A are deleted; Mutation site III is located after position 15274674 of the DNA of chromosome Gm07, and a base A is inserted at mutation site III; Mutation site IV is located at position 2423098 of chromosome DNA Gm08, and a base G is missing at mutation site IV.

2. The soybean ethylene synthesis key gene mutant according to claim 1 NKea-3 , characterized in that, The mutation site I is located above the mutation sequence I, and the nucleotide sequence of the mutation sequence I is shown in SEQ ID NO.1; The mutation site II is located above the mutation sequence II, and the nucleotide sequence of the mutation sequence II is shown in SEQ ID NO.2; The mutation site III is located above the mutation sequence III, and the nucleotide sequence of the mutation sequence III is shown in SEQ ID NO. 3; The mutation site IV is located above the mutation sequence IV, and the nucleotide sequence of the mutation sequence IV is shown in SEQ ID NO.

4.

3. The soybean ethylene synthesis key gene mutant according to claim 1 NKea-3, It is characterized in that the mutation sequence I encodes mutant protein I, whose amino acid sequence is shown in SEQ ID NO.5; the mutation sequence II encodes mutant protein II, whose amino acid sequence is shown in SEQ ID NO.6; the mutation sequence III encodes mutant protein III, whose amino acid sequence is shown in SEQ ID NO.7; and the mutation sequence IV encodes mutant protein IV, whose amino acid sequence is shown in SEQ ID NO.

8.

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

5. The soybean ethylene synthesis key gene mutant according to claim 4 NKea-3 The preparation method is characterized in that Step S100 includes the following steps: S110, using pCBC-DT1T2 as a template, two rounds of PCR amplification were performed with primers 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-3 gene editing vectors.

6. A mutant of a key gene for soybean ethylene synthesis NKea-3 The application is characterized in that The soybean ethylene synthesis key gene mutant according to any one of claims 1 to 3 NKea-3 The application includes improving the alkali stress resistance and / or drought stress resistance of crops.

7. The soybean ethylene synthesis key gene mutant according to claim 6 NKea-3 The application is characterized in that Improving the alkali stress resistance and / or drought stress resistance of crops includes breeding improved varieties of crops.

8. The soybean ethylene synthesis key gene mutant according to claim 6 NKea-3 The application is characterized in that Improving alkaline stress resistance and / or drought stress resistance in crop plants includes the production of hybrids of the crop plants.

9. The soybean ethylene synthesis key gene mutant according to claim 6 NKea-3 The application is characterized in that The crops are selected from one or more of leguminous crops, rice, corn, wheat, tomato, pineapple, cucumber, potato, sugarcane and cotton.

10. The soybean ethylene synthesis key gene mutant according to claim 9 NKea-3 The application is characterized in that The leguminous crops are selected from one or more of soybean, broad bean, pea, mung bean, red bean, pea, pigeon pea and chickpea.

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