Application of soybean GmKAO gene in improving drought resistance of soybean

Abstract

The application discloses application of a soybean GmKAO gene in improving drought resistance of soybean, and the improvement of the drought resistance of the soybean is realized by any of the following modes: simultaneously causing deletion of 120bp-124bp of a GmKAO1 gene and 105bp-110bp of a GmKAO2 gene; simultaneously causing deletion of 118bp-122bp of the GmKAO1 gene and 105bp-110bp of the GmKAO2 gene or simultaneously causing deletion of 120bp-124bp of the GmKAO1 gene and 109bp-112bp of the GmKAO2 gene. By deleting part of bases of the GmKAO1 gene and the GmKAO2 gene, survival rates, soil water content in a drought stress process, leaf relative water content and proline content of the obtained mutant strains are all significantly higher than those of wild plants TL1, and it is indicated that the GmKAO gene can improve the drought resistance of the soybean.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of crop genetic breeding, and particularly relates to application of a soybean GmKAO gene in improving drought resistance of soybean. BACKGROUND

[0002] In the past decade, with global warming, population growth, increasing demand for agricultural water, and decreasing availability of fresh water, the impact of drought on agricultural production has been further exacerbated. As an important food crop, soybean is an important source of edible soybean protein and oil in the world, and its water consumption during the whole growth period is large and the root system is not developed, which is extremely sensitive to drought. The annual yield reduction caused by drought is more than 40%, therefore, how to cope with agricultural drought is a prominent problem faced by the soybean industry.

[0003] Due to the complexity of soybean stress resistance traits and the diversity of yield influencing factors, the improvement of soybean stress resistance traits faces great challenges, therefore, focusing on the response and defense of soybean to drought stress, comprehensively understanding and mastering the response mechanism and defense mechanism of soybean to drought at the physiological, biochemical and molecular genetic levels has important and far-reaching theoretical and practical significance for breeding new drought-resistant soybean varieties, improving the stress resistance and productivity of soybean, and solving the problem of food security.

[0004] Soybean is a diploid species evolved from an ancient tetraploid, with a highly repetitive genome, in which about 75% of the predicted genes have multiple copies. Due to the complex structure of the soybean genome and the low genetic diversity in domesticated varieties, only a few genes that regulate drought stress in soybean have been cloned and identified. For example, the transcription factor GmWRKY54 is involved in drought stress regulation by activating the expression of Abscisic acid (ABA) receptor PYL8 (Pyrabactin resistance 1-like protein) and SnRK2 (SNF1-related protein kinase 2) kinases. Overexpression of GmWRKY54 in plants results in leaf stomatal closure, reduced water loss, and increased drought resistance (Zhou et al. 2008, Wei et al. 2019). The CCC class zinc finger protein family member GmZF351 promotes leaf stomatal closure and increases drought resistance in soybean by activating the expression of GmSnRK (Wei et al. 2023). A peroxidase GmPrx16 was identified through genome-wide association study (GWAS) of soybean field drought resistance index in response to drought stress. Overexpression of GmPrx16 significantly increased the peroxidase activity of soybean leaves and reduced the content of reactive oxygen species, thereby improving the drought resistance of soybean (Zhang et al. 2024). The above studies mainly focus on the mechanism of abscisic acid-mediated drought stress response.

[0005] The key enzyme in the biosynthesis of gibberellins (GA), ent-kaurenoic acid oxidase (KAO), is a subfamily CYP88A cytochrome P450 monooxygenase that catalyzes the conversion of ent-kaurenoic acid (KA) to GA 12 , providing a precursor for the synthesis of biologically active GAs. Recent studies have shown that KAO plays an important role in the growth and development of soybean. There are two KAO genes in Arabidopsis, which are highly expressed in developing organs such as germinating seeds and inflorescences. Loss of function of Arabidopsis KAO genes reduces the active content of GA in plants, resulting in a typical dwarf phenotype (Regnault et al. 2014). ZmGAD5 encodes a KAO protein, and loss of function of ZmGAD5 significantly increases the content of KAO, greatly reduces the content of GA 12 and active GA3, and ultimately leads to plant dwarfism, with significantly shortened stem node length, but without changing the development of plant roots and stem nodes (Li et al. 2023).

[0006] However, whether the soybean KAO is involved in the drought stress regulation of soybean needs to be further explored. SUMMARY

[0007] Based on this, the purpose of the present application is to provide the application of soybean GmKAO gene in improving the drought resistance of soybean.

[0008] The specific technical solutions for achieving the above-mentioned purposes include the following.

[0009] In the first aspect of the present application, the application of soybean GmKAO gene in improving the drought resistance of soybean is provided, characterized in that the improvement of the drought resistance of soybean is realized by any of the following ways:

[0010] (1) making the 120bp-124bp of the GmKAO1 gene with the sequence shown in SEQ ID NO: 1 and the 105bp-110bp of the GmKAO2 gene with the sequence shown in SEQ ID NO: 2 simultaneously deleted;

[0011] (2) making the 118bp-122bp of the GmKAO1 gene with the sequence shown in SEQ ID NO: 1 and the 105bp-110bp of the GmKAO2 gene with the sequence shown in SEQ ID NO: 2 simultaneously deleted;

[0012] (3) making the 120bp-124bp of the GmKAO1 gene with the sequence shown in SEQ ID NO: 1 and the 109bp-112bp of the GmKAO2 gene with the sequence shown in SEQ ID NO: 2 simultaneously deleted.

[0013] In the second aspect of the present application, the application of soybean GmKAO gene in cultivating drought-resistant soybean varieties is provided.

[0014] In the third aspect of the present application, the application of the knock-out vector of soybean GmKAO gene in improving the drought resistance of soybean or cultivating drought-resistant soybean varieties is provided.

[0015] In the fourth aspect of the present application, the application of the engineering bacteria transformed with the knock-out vector of soybean GmKAO gene in improving the drought resistance of soybean or cultivating drought-resistant soybean varieties is provided.

[0016] In a fifth aspect of the present application, a method for improving drought resistance of soybean is provided, comprising the following steps: simultaneously deleting 120bp-124bp of the GmKAO1 gene shown in SEQ ID NO:1 and 105bp-110bp of the GmKAO2 gene shown in SEQ ID NO:2, simultaneously deleting 118bp-122bp of the GmKAO1 gene shown in SEQ ID NO:1 and 105bp-110bp of the GmKAO2 gene shown in SEQ ID NO:2, or simultaneously deleting 120bp-124bp of the GmKAO1 gene shown in SEQ ID NO:1 and 109bp-112bp of the GmKAO2 gene shown in SEQ ID NO:2.

[0017] The inventors of the present application found that, by simultaneously knocking out the GmKAO1 gene and the GmKAO2 gene of a soybean plant, and deleting part of the bases of the GmKAO1 gene and the GmKAO2 gene, the survival rate, the soil water content during drought stress, the leaf relative water content, and the proline content of the obtained mutant plant are all significantly higher than those of the wild plant TL1; the anthocyanin, propylene glycol MDA content, and H2O2 content are significantly lower than those of the wild plant TL1, indicating that the GmKAO gene negatively regulates the drought resistance of soybean. The discovery of the new function of the soybean GmKAO gene provides a new gene target and resource for genetic breeding of drought-resistant soybean. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 FIG. 1 is a structural diagram of the soybean GmKAO gene knockout vector constructed in Embodiment 1 of the present application, wherein the size of SgRNA is 83bp+19bp+1bp in the primer design process = 103bp.

[0019] Figure 2 FIG. 2 is the target sequencing result of the soybean GmKAO gene knockout mutant strain in Embodiment 2 of the present application, wherein TL1 is a wild-type soybean, and gmkao-1, gmkao-2, and gmkao-3 are GmKAO gene knockout mutants.

[0020] Figure 3 FIG. 3 is the related result of drought tolerance identification of the soybean GmKAO gene knockout strain in Embodiment 3 of the present application, wherein TL1 is a wild-type soybean, and gmkao-1 and gmkao-3 are GmKAO gene knockout strains. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of the present application, the present application will be described more fully below. The present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0022] Unless otherwise defined, 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 application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0023] Unless otherwise indicated, all experiments were carried out in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning: A Laboratory Manual (2013), or as suggested by the manufacturer's instructions.

[0024] In some embodiments of the present application, the application of soybean GmKAO gene in improving drought resistance of soybean is disclosed, and the improvement of drought resistance of soybean is achieved by any of the following ways:

[0025] (1) deleting 120bp-124bp of GmKAO1 gene with sequence as shown in SEQ ID NO: 1 and deleting 105bp-110bp of GmKAO2 gene with sequence as shown in SEQ ID NO: 2;

[0026] (2) deleting 118bp-122bp of GmKAO1 gene with sequence as shown in SEQ ID NO: 1 and deleting 105bp-110bp of GmKAO2 gene with sequence as shown in SEQ ID NO: 2;

[0027] (3) deleting 120bp-124bp of GmKAO1 gene with sequence as shown in SEQ ID NO: 1 and deleting 109bp-112bp of GmKAO2 gene with sequence as shown in SEQ ID NO: 2.

[0028] In some embodiments of the present application, the application of soybean GmKAO gene in breeding drought-resistant soybean varieties is disclosed.

[0029] In some other embodiments of the present application, the application of knock-out vector of soybean GmKAO gene in improving drought resistance of soybean is disclosed, and the improvement of drought resistance of soybean is achieved by any of the following ways:

[0030] (1) deleting 120bp-124bp of GmKAO1 gene with sequence as shown in SEQ ID NO: 1 and deleting 105bp-110bp of GmKAO2 gene with sequence as shown in SEQ ID NO: 2 at the same time;

[0031] (2) simultaneously deleting 118bp-122bp of the GmKAO1 gene with sequence as shown in SEQ ID NO: 1 and 105bp-110bp of the GmKAO2 gene with sequence as shown in SEQ ID NO: 2;

[0032] (3) simultaneously deleting 120bp-124bp of the GmKAO1 gene with sequence as shown in SEQ ID NO: 1 and 109bp-112bp of the GmKAO2 gene with sequence as shown in SEQ ID NO: 2.

[0033] In some embodiments, the sequence of the sgRNA of the knockout vector is as shown in SEQ ID NO: 10.

[0034] In some embodiments, the knockout vector of the soybean GmKAO gene is constructed by the following method:

[0035] S1. amplifying a GmU6 promoter fragment using soybean leaf DNA as a template and SEQ ID NO: 5 and SEQ ID NO: 6 as primers;

[0036] S2. amplifying a fragment containing sgRNA as shown in SEQ ID NO: 10 using SEQ ID NO: 7 as a template and SEQ ID NO: 8 and SEQ ID NO: 9 as primers;

[0037] S3. amplifying a GmU6-sgRNA fragment using the GmU6 promoter fragment and the fragment containing sgRNA as shown in SEQ ID NO: 10 as templates and SEQ ID NO: 8 and SEQ ID NO: 9 as primers;

[0038] S4. connecting the GmU6-sgRNA fragment and the cleaved CRISPR vector, and obtaining the knockout vector.

[0039] In some embodiments of the present application, the application of the knockout vector of the soybean GmKAO gene in breeding drought-resistant soybean varieties is disclosed.

[0040] In some embodiments of the present application, the application of the engineered bacteria transformed with the knockout vector of the soybean GmKAO gene in improving the drought resistance of soybean is disclosed.

[0041] In some embodiments of the present application, the application of the engineered bacteria transformed with the knockout vector of the soybean GmKAO gene in breeding drought-resistant soybean varieties is disclosed.

[0042] In some embodiments of the present application, a method for improving drought resistance of soybean is disclosed, which comprises the following steps: simultaneously deleting 120bp-124bp of GmKAO1 gene shown in SEQ ID NO: 1 and 105bp-110bp of GmKAO2 gene shown in SEQ ID NO: 2, or simultaneously deleting 118bp-122bp of GmKAO1 gene shown in SEQ ID NO: 1 and 105bp-110bp of GmKAO2 gene shown in SEQ ID NO: 2, or simultaneously deleting 120bp-124bp of GmKAO1 gene shown in SEQ ID NO: 1 and 109bp-112bp of GmKAO2 gene shown in SEQ ID NO: 2.

[0043] In some embodiments, the deletion is achieved by the following steps: infecting the cotyledon node of soybean with the engineering bacteria of the knock-out vector of the transformed soybean GmKAO gene, and then obtaining the tissue culture seedlings of soybean, and finally obtaining the transgenic lines (i.e. the knock-out lines of soybean GmKAO gene).

[0044] The present application will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Example 1: Construction of the vector for knocking out soybean GmKAO gene

[0046] In this example, the GmU6 promoter and sgRNA containing 19bp target site sequence (TGGCTCTATGAATCCAAAT) were constructed into the CRISPR vector through the restriction enzyme XbaI site by using the Infusion system (Novagen), and the vector for simultaneously knocking out soybean GmKAO1 gene and GmKAO2 gene (named as CRISPR-GmKAO12) was obtained, wherein the CDS sequence of soybean GmKAO1 gene is shown in SEQ ID NO: 1, the amino acid sequence of the encoded protein is shown in SEQ ID NO: 3, the CDS sequence of soybean GmKAO2 gene is shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 4.

[0047] Nucleotide sequence of GmKAO1 (SEQ ID NO: 1):

[0048] ATGATAATGATGATGATGATGTGTTCCATGTGGATGTGGGTTGTCCTTGTGGCC

[0049] ATTGCTGGTGCCCTTTTAGTCCTAAGATCTATCCTCAAGAATGTAAATTGGTGG

[0050] CTCTATGAATCCAAATTGGGTGTGAAGCAGTACTCTTTGCCACCAGGTGACAT

[0051] GGGATGGCCCTTCATTGGCAACATGTGGTCCTTTCTCAGTGCTTTCAAGTCCAA

[0052] GGACCCTGATTCCTTCATCTCCTCCTTTGTCTCCAGATTTGGAAGAACTGGAAT

[0053] GTACAAGACCATGATGTTTGGAAATCCAAGTATAATTGTGACAACACCTGAAA

[0054] TATGCAAAAGGGTGCTTACAGATGACGATAAATTCACACCTGGTTGGCCTCAA

[0055] TCTACTATAGAGCTCATTGGAAAGAGGTCATTTATTTCAATGTCTTATGAAGAA

[0056] CATAAGCGCCTTAGGCGTTTGACATCCTCTTCAATCAATGGCATGGAAGCACT

[0057] GTCCCTCTACTTGACATATATTGAAAAAAATGTGAAATCTTCCTTGGAGAAAT

[0058] GGGCCAACATGGGACAAATTGAGTTCTTAACTGAGATCAGGAAGCTTACTTTC

[0059] AAAATCATTATGCATATTTTCCTTAGCTCAGAGAGTGAACATGTTATGGAGGC

[0060] TTTGGAGAGGGAATACACAGCACTTAATCATGGAGTTAGAGCCATGTGTATTA

[0061] ATATTCCCGGATTTGCATACCACAAAGCATTCAAGGCAAGGAAAAATCTAGTG

[0062] GCCATATTTCAATCTATTGTGGACGAGAGAAGAAACTTAAGGAAGGGCTATCT

[0063] GCCTGGAAAAGCCAAAGATATGATGGATGCTCTGATAGATCTTGAAGATGATG

[0064] AAAGAAAGTTGAGTGATGAGGACATCATTGACATCATGTTGATGTACTTGAAT

[0065] GCGGGCCACGAGTCTTCAGGACATATTACCATGTGGGCAACCTTCTTCCTGCA

[0066] AAAGCACCCGAATATCTCCAAAAGGCTAAGGCAGAACAAGAAGAAATAATA

[0067] AGGAGAAGGCCTTCAACACAGAAAGGGTTGACACTTAAGGAAGTTCGGGGAGA

[0068] TGGATTTTCTTTATAAGGTGATTGATGAAACATTGCGTGTGATTACATTCTCAC

[0069] TAGTGGTCTTTCGGGAGGCAAAAACTGATGTTAATATCAATGGATACACAGTT

[0070] CCAAAAGGTTGGAAAGTGCTTGTGTGGTTCAGATCAGTTCACCTTGATCCTGA

[0071] AATATTTCCTGATCCAAAGGAATTTAACCCTAATAGATGGAATAAAAGAGCACA

[0072] AGGCTGGAGAATTCCTTCCCTTTGGAGGAGGAAGTAGATTGTGTCCTGGGGAAT

[0073] GATCTTGCCAAGATGGAAATAGCAGTTTTTCTTCACCATTTCCTTCCTGAATTAC

[0074] CGATTTGAACAGCATAATCCTAATTGCCCTGTGAGATACTTGCCACATACAAG

[0075] GCCAATGGACAATTGCTTGGGAAGGGTCAGGAAATGTCCATCTACAACAACCT

[0076] AA

[0077] Amino acid sequence of GmKAO1 (SEQ ID NO: 3):

[0078] MIMMMMMCSMWMWVVLVAIAGALLVLRSILKNVNWWLYESKLGVKQYSLPP

[0079] GDMGWPFIGNMWSFLSAFKSKDPDSFISSFVSRFGRTGMYKTMMFGNPSIIVTTPEI

[0080] CKRVLTDDDKFTPGWPQSTIELIGKRSFISMSYEEHKRLRRLTSSSINGMEALSLYL

[0081] TYIEKNVKSSLEKWANMGQIEFLTEIRKLTFKIIMHIFLSSESEHVMEALEREYTAL

[0082] NHGVRAMCINIPGFAYHKAFKARKNLVAIFQSIVDERRNLRKGYLPGKAKDMMD

[0083] ALIDLEDDERKLSDEDIIDIMLMYLNAGHESSGHITMWATFFLQKHPEYLQKAKAE

[0084] QEEIIRRRPSTQKGLTLKEVREMDFLYKVIDETLRVITFSLVVFREAKTDVNINGYT

[0085] VPKGWKVLVWFRSVHLDPEIFPDPKEFNPNRWNKEHKAGEFLPFGGGSRLCPGNDLAKMEIAVFLHHFLLNYRFEQHNPNCPVRYLPHTRPMDNCLGRVRKCPSTTT* Nucleotide sequence of GmKAO2 (SEQ ID NO: 2):

[0086] ATGATGGAGATGGATTCCATGTGTATGTGGGTGGTCCTTGTGGCCATTGCTGGT

[0087] GCTCTTTTAGTCCTAAGATCTATGCTCAAGAATGTAAATTGGTGGCTCTATGAA

[0088] TCCAAATTGGGTGTGAAACAGTACTCTTTGCCCCCAGGTGACATGGGATGGCC

[0089] CTTCATTGGCAACATGTGGTCCTTCCTCAGAGCTTTCAAGTCCAAGGACCCTGA

[0090] TTCCTTCATCTCCTCCTTTGTCTCCAGATATGGAAGAACTGGAATGTACAAGAC

[0091] CTTGATGTTTGGAAATCCAAGTGTAATTGTGACAACACCTGAAACATGCAAAA

[0092] GGGTGCTTACAGACGATGATAAATTCACAACTGGTTGGCCTCAATCTACTATA

[0093] GAGCTCATTGGAAAGAGGTCATTTATTTCAATGTCTTATGAAGAACATAAACG

[0094] CCTTAGGCGTTTGACATCCTCTTCAATCAATGGCATGGAATCACTGTCCCTCTA

[0095] CTTGACATATATTGAAGAAAATGTGAAAAATTCATTGGAGAAATGGGCCAACA

[0096] TGGGACAAATTGAGTTCTTAACTGAGATCAGGAAGCTTACTTTCAAAATCATC

[0097] ATGCATATTTTCCTTAGCTCAGAAAGTGAACCTGTTATGGAGGCTTTGGAGAG

[0098] GGAATACACAGCACTTAATCATGGAGTTAGAGCCATGTGTATTAATATTCCCG

[0099] GATTTGCATACCACAAAGCATTCAAGGCAAGGAAAAATCTAGTGGCCATATTT

[0100] CAATCTATTGTGGATGAGAGAAGAAACTTAAGGAAGGGATATCTGCCAGGAA

[0101] AAGCCAAAGATATGATGGATGCTCTGATAGATGTTGAAGATGATGATGGAAG

[0102] AAAGTTGAGTGATGAGGACATCATTGACATTATGTTGATGTACTTGAATGCGG

[0103] GCCATGAGTCTTCAGGACATATTACCATGTGGGCAACCTTTTTCCTGCAGAAG

[0104] CACCCAGAATATCTCCAAAAGGCTAAGGCAGAACAAGAAGAAATAATAAGGA

[0105] GAAGGCCTCCAACACAGAAAGGGTTAACACTTAAGGAAGTCCGGGAGATGGA

[0106] TTTTCTTTACAAGGTGATTGATGAAACATTGCGTGTGATTACATTCTCACTAGT

[0107] GGTCTTTCGGGAGGCAAAATCTGATGTCAATATCAATGGATACACAATTCCAA

[0108] AAGGTTGGAAAGCGCTTGTGTGGTTCAGATCAGTTCACCTTGATCCTGAAATA

[0109] TATCCTAATCCAAAGGAATTTAACCCTTATAGATGGAATAAAGAACACAAGGC

[0110] CGGAGAATTCCTTCCCTTTGGAGGAGGAAGTAGATTGTGTCCGGGGAACGATC

[0111] TTGCCAAGATGGAAATAGCAGTTTTTCTTCACCATTTCCTTCTGAATTACCGAT

[0112] TTGAACAGCATAATCCTAATTGCCCTGTGAGATACTTACCACATACAAGGCCC

[0113] ATGGACAATTGCTTGGGAAGGGTCAGGAAATGTTCATCTACAACAACCTAAGmKAO2 of the amino acid sequence (SEQ ID NO: 4):

[0114] MMEMDSMCMWVVLVAIAGALLVLRSMLKNVNWWLYESKLGVKQYSLPPGDM

[0115] GWPFIGNMWSFLRAFKSKDPDSFISSFVSRYGRTGMYKTLMFGNPSVIVTTPETCK

[0116] RVLTDDDKFTTGWPQSTIELIGKRSFISMSYEEHKRLRRLTSSSINGMESLSLYLTYI

[0117] EENVKNSLEKWANMGQIEFLTEIRKLTFKIIMHIFLSSESEPVMEALEREYTALNHG

[0118] VRAMCINIPGFAYHKAFKARKNLVAIFQSIVDERRNLRKGYLPGKAKDMMDALID

[0119] VEDDDGRKLSDEDIIDIMLMYLNAGHESSGHITMWATFFLQKHPEYLQKAKAEQE

[0120] EIIRRRPPTQKGLTLKEVREMDFLYKVIDETLRVITFSLVVFREAKSDVNINGYTIPK

[0121] GWKALVWFRSVHLDPEIYPNPKEFNPYRWNKEHKAGEFLPFGGGSRLCPGNDLAKMEIAVFLHHFLLNYRFEQHNPNCPVRYLPHTRPMDNCLGRVRKCSSTTT*

[0122] The method for constructing a gene knockout vector specifically comprises the following steps:

[0123] 1. Take soybean William 82 seeds, plant in nutrient soil, grow for 15 days under long day conditions, and take the first trifoliate leaf of soybean to extract DNA. Among them, the method for extracting DNA is as follows:

[0124] (1) Take an appropriate amount of tender soybean leaves and put them into a 1.5 mL RNase-free centrifuge tube containing a sample punching steel ball, and quickly place them in liquid nitrogen. Grind the sample with a sample puncher or a mortar under a frozen state, add 500 μL of 65°C preheated CTAB solution, mix well by vortex, and place in a 65°C drying oven for 30 min.

[0125] (2) Take the sample from the drying oven and cool it down, add 500 μL of 25:24:1 Tris-saturated phenol / chloroform / isoamyl alcohol solution (must be cooled to room temperature before adding the solution), mix well by inverting or vortexing, and centrifuge at a constant speed of 12,000 rpm for 15 min at room temperature. Absorb the supernatant into a new centrifuge tube.

[0126] (3) Add 500 μL of 24:1 chloroform / isoamyl alcohol solution to a new 1.5 mL centrifuge tube and mix well by inverting or vortexing, and centrifuge at a constant speed of 12,000 rpm for 10 min at room temperature. Slowly absorb 500 μL of supernatant into a new centrifuge tube.

[0127] (4) Add 500 μL of isopropanol at room temperature, mix well by inverting or vortexing, and place at -20°C for a period of time or at -80°C overnight, and centrifuge at a constant speed of 12,000 rpm for 10 min at room temperature.

[0128] (5) Discard the supernatant, add 1 mL of 75% ethanol, mix well by inverting and centrifuge at 12,000 rpm for 10 min at room temperature, remove the supernatant again, and place at room temperature for 5 min. Remove the excess ethanol with a gun head, and place at room temperature for 20 min. Add 40 μL of ultrapure water to dissolve, ready for later use.

[0129] 2. Take soybean leaf DNA as a template, GmU6-XbaI-F (SEQ ID NO: 5) and GmU6-R (SEQ ID NO: 6) as primers, and amplify GmU6 promoter fragment. The PCR amplification reaction system and reaction program are shown in Table 1 and Table 2.

[0130] SEQ ID NO: 5:

[0131] GGAAGCTTAGGCCTTCTAGAAAAATAAATGGTAAAATGTC

[0132] SEQ ID NO: 6: CAATCCATGTGGTGGCACAT

[0133] The SgRNA fragment (TGGCTCTATGAATCCAAAT, SEQ ID NO: 10) containing the target site (19bp) of Gmkao1 and Gmkao2 genes was amplified with SEQ ID NO: 7 as the template, Gmkao-F (SEQ ID NO: 8, the underlined sequences are the target site sequences of Gmkao1 and Gmkao2 genes) and sgRNA-XbaI-R (SEQ ID NO: 9) as primers, and the PCR amplification reaction system and reaction procedure are shown in Table 1 and Table 2.

[0134] SEQ ID NO: 7:

[0135] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT

[0136] TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

[0137] SEQ ID NO: 8:

[0138] TGTGCCACCACATGGATTG TGGCTCTATGAATCCAAAT GTTTTAGAGCTAG

[0139] AAATAGC

[0140] SEQ ID NO: 9: GCAACGCGTTCTAGAAAAAAAAGCACCGACTCGGTGCCAC

[0141] Table 1

[0142]

[0143] Table 2

[0144] 95℃ 2 min 95℃ 15s 57-62℃ 15s 72℃ 1 min / kb 2-4 cycles 35-36 72℃ 5 min 4℃ Long term storage

[0145] 3, the GmU6 promoter fragment obtained in step 2 and the SgRNA fragment containing the 19bp target site sequence were used as templates, Gmkao-F (SEQ ID NO: 8) and sgRNA-XbaI-R (SEQ ID NO: 9) were used as primers, and the GmU6-SgRNA fragment containing the target site (19bp) of Gmkao1 and Gmkao2 genes was amplified, and the PCR amplification reaction system and reaction procedure are shown in Table 1 (except that the templates are different, and other conditions are the same) and Table 2.

[0146] 4, the CRISPR vector was digested with restriction enzyme XbaI (the enzyme digestion reaction system is shown in Table 3).

[0147] Table 3

[0148]

[0149] 5. Perform gel electrophoresis on the PCR products from step 3 and the digested vector from step 4 to determine the size of the target band and remove it (use the Axygen gel extraction kit to recover various corresponding products; follow the Axygen instructions for detailed experimental procedures). Place the target band in a 1.5 mL centrifuge tube and ligate at 37°C for 30 min (ligation reaction shown in Table 4) to obtain the CRISPR vector with simultaneous knockout of the soybean GmKAO1 and GmKAO2 genes (named CRISPR-GmKAO12, structure as shown). Figure 1 (As shown), store at 4℃.

[0150] Table 4

[0151]

[0152] Example 2: Obtaining soybean GmKAO gene knockout plants

[0153] This embodiment uses Agrobacterium EHA105-mediated genetic transformation to transform the soybean GmKAO1 and GmKAO2 gene knockout CRISPR vector constructed in Example 1 into soybean cotyledonary nodes, obtaining soybean plants with simultaneous GmKAO1 and GmKAO2 gene knockout, which include the following steps:

[0154] 1. Preparation of Agrobacterium competent cells

[0155] Single colonies of Agrobacterium tumefaciens EHA105 were picked and placed in 5 mL of LB broth containing the corresponding antibiotic (EHA105 resistance: 100 μg / mL rifampin). The cultures were incubated overnight at 28°C. 500 μL of the overnight culture was then inoculated into 50 mL of LB broth containing the corresponding antibiotic and incubated at 28°C until OD500 reached. 600 Approximately 0.5; place on ice for 30 min; centrifuge at 5,000 rpm for 10 min at 4 °C; resuspend Agrobacterium cells in 15 mL of pre-chilled 10 mM CaCl2; centrifuge at 5,000 rpm for 10 min at 4 °C; resuspend the precipitate in 2 mL of pre-chilled 10 mM CaCl2; aliquot 100 μL / tube on ice; flash freeze in liquid nitrogen; store at -80 °C.

[0156] 2. Electroporation transformation of Agrobacterium

[0157] In 50 μL of Agrobacterium competence, 3 μL of plasmid (i.e., soybean GmKAO gene knockout vector constructed in Example 1) was added, mixed and homogenized by a sterile gun head, and placed on ice for 30 min. During this period, the super-clean workbench of the electroporation cup was dried and pre-cooled. The competence was transferred to the electroporation cup on ice, and the electroporation transformation was prepared. The BIO-RAD transformation instrument (Gene PulserXcell Electroporation System) Bacterial electroporation transformation was used. After electroporation, 700 μL of antibiotic-free LB liquid medium was added to the super-clean bench to recover the bacterial liquid, and the recovered bacterial liquid was incubated at 28°C on a shaker at 200 rpm for 2 h. The bacterial liquid was collected by centrifugation and plated. After 2-3 days of culture, the Agrobacterium was grown, and the Agrobacterium monoclonal was screened according to the method of screening positive E. coli monoclonal.

[0158] 3. Agrobacterium infection of soybean cotyledon node

[0159] The co-culture solid / liquid medium, induction solid / liquid medium, elongation solid medium, and rooting medium in this step are all conventional commercially available media.

[0160] (1) The positive Agrobacterium monoclonal screened in step 2 was cultured in a 10 mL test tube overnight.

[0161] (2) 100 mL of Bleach + 4 mL of concentrated hydrochloric acid was used to sterilize soybeans in a sealed container for 16-18 hours. The soybeans were placed in a sterilized super-clean bench for 30 minutes, and the chlorine gas was blown away.

[0162] (3) Prepare co-culture solid / liquid medium, water, filter paper, and Erlenmeyer flask, and sterilize at 121°C for 20 min. Sterilized water was used to soak the beans for about 8 hours (usually, the beans were placed in a 28°C dark incubator for about 8 hours at 6-10 pm, and the beans were not soaked for too long). Meanwhile, 50 mL of Agrobacterium was shaken in an Erlenmeyer flask for infection.

[0163] (4) The soaked beans were cut in half with a scalpel, and the excess hypocotyl part was removed from 3-4 mm of the hypocotyl. The blade was gently drawn 5-7 times at the hypocotyl node, with a depth of 0.5 mm. After cutting, the bean pieces were placed in a conical flask with water.

[0164] (5) When the OD 600 of Agrobacterium was 0.5-0.7, centrifugation was performed at 4,000 rpm for 10 min, the supernatant was removed, and an appropriate amount of co-culture liquid medium was added, vortexed to mix, and the OD 600 of the bacterial liquid was adjusted to 0.6.

[0165] (6) After the beans are cut, the water in the conical flask is removed, and the bacteria solution prepared with co-culture liquid medium is added. Shake 2-3 times, and infect for 30 minutes, with appropriate shaking in between. Meanwhile, sterilized filter paper is laid on the co-culture solid medium, with one filter paper for one co-culture solid medium. After the infection is completed, the bacteria solution is removed, and the infected beans are placed on the empty culture dish containing several layers of filter paper, and the excess bacteria solution is absorbed. Then, the beans are evenly placed on the co-culture solid medium with filter paper laid thereon, and cultured in the dark at 28°C for 3 days. 20-30 beans can be laid on one co-culture solid medium. Too many beans laid on one co-culture solid medium can cause poor growth of the beans and cross contamination.

[0166] (7) Prepare the induction solid / liquid medium, water, filter paper and conical flask, and sterilize them at 121°C for 20 minutes. Remove the bean embryos and the excess hypocotyls from the beans that have been co-cultured for 3 days, and retain 3-4 mm of the hypocotyls. Wash the hypocotyls with water for 4-5 times, and wash with the induction liquid medium for 4-5 times. Place the hypocotyls on the empty culture dish containing filter paper, and absorb the water. Then, insert the hypocotyls into the induction solid medium with the hypocotyls upward, and culture under light for 10 days.

[0167] (8) After 10 days, remove the larger embryos at the base of the beans, and gently scratch 5-7 places on the hypocotyls. Insert the hypocotyls into the new induction solid medium with the hypocotyls upward, and culture under light for 10 days. Repeat twice.

[0168] (9) Prepare the elongation solid medium, and sterilize it at 121°C for 20 minutes. After 3 times of induction culture, most of the beans grow callus with embryos. Remove the beans, retain the callus, remove the black tissue on the surface of the callus, and place the remaining callus into the elongation medium, and culture under light for 10 days. Repeat three times.

[0169] (10) During the replacement of the elongation medium, when the embryos of the callus grow to a length of more than 3 cm, cut them from the callus, and place them into the rooting medium, and culture under light for 10-14 days. After the seedlings grow roots, transplant them into the soil, and acclimate them for about 7 days, to obtain the T0 generation of transgenic seedlings.

[0170] 4. Identification of soybean GmKAO gene knockout lines

[0171] Take the leaves of the T0 and T1 generation of transgenic seedlings, extract DNA, and perform PCR amplification using primer pairs GmKAO1-F (SEQ ID NO: 11) and GmKAO1-R (SEQ ID NO: 12), and GmKAO2-F (SEQ ID NO: 13) and GmKAO2-R (SEQ ID NO: 14), respectively. The reaction volume is 50 μL (the reaction system is shown in Table 1, and the reaction procedure is shown in Table 2).

[0172] SEQ ID NO: 11: TCCCTTATAAATACACAGAA

[0173] SEQ ID NO: 12: AACTTAGACTCACAAGTCAC

[0174] SEQ ID NO: 13: GCCCCAGTTGAGTAGTAAGT

[0175] SEQ ID NO: 14: GAGGAGGATCATGGGAACTA

[0176] After PCR, 5 μL of the gel was taken to detect whether there was a specific band. The remaining 45 μL sample was sent to Genesee Biologics Co., Ltd. for first generation sequencing and compared with wild type genomic DNA to determine whether the soybean GmKAO gene knockout strain was a homozygous mutant. Finally, three homozygous mutant soybean GmKAO gene knockout strains (mutants) were obtained, named gmkao-1, gmkao-2 and gmkao-3, respectively. The sequencing results of the target points of the gene knockout strains are shown in Table 1. Figure 2 Figure 2 As can be seen, in the mutant gmkao-1, the GmKAO1 gene is deleted by 5 bp and the GmKAO2 gene is deleted by 6 bp; in the mutant gmkao-2, the GmKAO1 gene is deleted by 5 bp and the GmKAO2 gene is deleted by 6 bp; in the mutant gmkao-3, the GmKAO1 gene is deleted by 5 bp and the GmKAO2 gene is deleted by 4 bp.

[0177] Example 3 Identification of drought tolerance of soybean GmKAO gene knockout strains

[0178] Tianlong No. 1 (control TL) and soybean GmKAO gene knockout strains gmkao-1 and gmkao-3 were planted under long-day conditions (16 hours light / 8 hours dark) for 2 weeks, each material was planted separately in a small pot, and drought stress treatment was carried out by stopping water supply for two weeks, and then water supply was restored. Before stopping water supply (control), 6 days after stopping water supply and 10 days after restoring water supply, the properties of Tianlong No. 1 and the gene knockout strains gmkao-1 and gmkao-3 were observed (A in FIG. 1), and the drought tolerance was detected after 10 days of restoring water supply. Figure 3

[0179] The results show that the soil water content (B in FIG. 2), the relative water content of leaves (C in FIG. 2), the survival rate (D in FIG. 2), and the proline content (E in FIG. 2) of gmkao-1 and gmkao-3 plants during the drought stress process were lower than those of Tianlong No. 1 (control) and the difference was significant (P < 0.05). Figure 3 Figure 3 Figure 3 Figure 3 ​​​​​F) in Table 1 were significantly higher than that of TL1; the gmkao-1 and gmkao-3 plants accumulated anthocyanin (G) in Table 1 under drought stress Figure 3 E) in Table 1, malondialdehyde MDA content (G) in Table 1, and H2O2 content (H) in Table 1 were significantly lower than that of TL1. Figure 3 Figure 3

[0180] The results of the above examples show that the drought resistance of soybean can be improved by simultaneously knocking out and prematurely terminating the gibberellin synthesis genes GmKAO1 and GmKAO2, which proves that GmKAO1 and GmKAO2 negatively regulate the drought resistance of soybean, and the drought resistance of the gmkao-3 plant is significantly stronger than that of the gmkao-1 plant.

[0181] Each technical feature of the above-described examples can be combined arbitrarily, and to make the description concise, all possible combinations of each technical feature in the above examples are not described, however, as long as the combinations of the technical features do not contradict, they should be considered as the scope of the description.

[0182] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.​​

Claims

1. The application of soybean GmKAO gene knockout in improving soybean drought resistance, wherein the soybean GmKAO gene is the GmKAO1 gene with the sequence shown in SEQ ID NO:1 and the GmKAO2 gene with the sequence shown in SEQ ID NO:

2.

2. Application of soybean GmKAO gene knockout in breeding drought-resistant soybean varieties, wherein the soybean GmKAO gene is the GmKAO1 gene with the sequence shown in SEQ ID NO:1 and the GmKAO2 gene with the sequence shown in SEQ ID NO:

2.

3. Application of soybean GmKAO gene knockout vector in improving soybean drought resistance, wherein the soybean GmKAO gene is the GmKAO1 gene with the sequence shown in SEQ ID NO:1 and the GmKAO2 gene with the sequence shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The sequence of the sgRNA of the knockout vector is shown in SEQ ID NO:

10.

5. The application according to claim 4, characterized in that, The soybean GmKAO gene knockout vector was constructed using the following method: S1. Using soybean leaf DNA as a template, and SEQ ID NO:5 and SEQ ID NO:6 as primers, the GmU6 promoter fragment was amplified; S2. Using SEQ ID NO:7 as a template and SEQ ID NO:8 and SEQ ID NO:9 as primers, amplify the fragment containing the sgRNA shown in SEQ ID NO:10; S3. Using the GmU6 promoter fragment and the fragment containing the sgRNA shown in SEQ ID NO:10 as templates, and SEQ ID NO:8 and SEQ ID NO:9 as primers, the GmU6-sgRNA fragment was amplified. S4. Ligate the GmU6-sgRNA fragment with the enzyme-digested CRISPR vector to obtain the final product.

6. Application of soybean GmKAO gene knockout vector in breeding drought-resistant soybean varieties, wherein the soybean GmKAO gene is the GmKAO1 gene with the sequence shown in SEQ ID NO:1 and the GmKAO2 gene with the sequence shown in SEQ ID NO:

2.

7. Application of engineered bacteria transforming soybean GmKAO gene knockout vector in improving soybean drought resistance, wherein the soybean GmKAO gene is the GmKAO1 gene with sequence as shown in SEQ ID NO:1 and the GmKAO2 gene with sequence as shown in SEQ ID NO:

2.

8. Application of engineered bacteria transforming soybean GmKAO gene knockout vector in the breeding of drought-resistant soybean varieties, wherein the soybean GmKAO gene is the GmKAO1 gene with the sequence shown in SEQ ID NO:1 and the GmKAO2 gene with the sequence shown in SEQ ID NO:

2.

9. A method for improving the drought resistance of soybeans, characterized in that, It includes the following steps: The soybean GmKAO gene was knocked out in soybean plants. The soybean GmKAO gene is the GmKAO1 gene with the sequence shown in SEQ ID NO:1 and the GmKAO2 gene with the sequence shown in SEQ ID NO:

2.

10. The method for improving the drought resistance of soybeans according to claim 9, characterized in that, The knockout is achieved through the following steps: the engineered bacteria that transform the soybean GmKAO gene knockout vector infect the soybean cotyledon nodes, and then soybean tissue culture is performed to obtain tissue culture seedlings, and then transgenic lines are obtained.

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