Application of soybean GmKAO gene in improving drought resistance of soybeans

Abstract

The invention discloses an application of a soybean GmKAO gene in improving the drought resistance of soybeans. The improvement of the drought resistance of the soybeans is realized by any one of the following ways: simultaneously deleting 120bp to 124bp of a GmKAO1 gene and 105bp to 110bp of a GmKAO2 gene; and the 118bp to 122bp of the GmKAO1 gene and the 105bp to 110bp of the GmKAO2 gene are subjected to deletion at the same time, or the 120bp to 124bp of the GmKAO1 gene and the 109bp to 112bp of the GmKAO2 gene are subjected to deletion at the same time. The survival rate, the soil water content, the leaf relative water content and the proline content of the obtained mutant strain in the drought stress process are obviously higher than those of a wild plant TL1 by deleting part of basic groups of the GmKAO1 gene and the GmKAO2 gene, and the result shows that the GmKAO gene can improve the drought resistance of soybeans.

Description

Technical Field

[0001] The invention belongs to the technical field of crop genetic breeding, and in particular relates to an application of soybean GmKAO gene in improving soybean drought resistance. Background Art

[0002] In the past decade, with global warming, population growth, increased demand for agricultural water, and reduced available fresh water, the impact of drought on agricultural production has been further exacerbated. As an important food crop, soybeans are an important source of edible soybean protein and oil in the world. They consume a lot of water during their entire growth period and have an underdeveloped root system. They are extremely sensitive to drought, and the annual production reduction caused by drought is more than 40%. Therefore, how to deal with agricultural drought is a prominent problem facing the soybean seed industry.

[0003] Due to the complexity of soybean stress resistance traits and the diversity of factors affecting yield, the improvement of soybean stress resistance traits faces huge challenges. Therefore, focusing on the research on soybean response and defense to drought stress, and comprehensively understanding and mastering the response mechanism and resistance 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 soybean stress resistance and productivity, and solving food security issues.

[0004] As a diploid soybean evolved from ancient tetraploid, soybean has a highly repetitive genome, of which about 75% of 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 regulating soybean drought stress have been cloned and identified. For example, the transcription factor GmWRKY54 participates 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) kinase. GmWRKY54 overexpression plants show leaf stomatal closure, reduced water loss and improved drought resistance phenotypes (Zhou et al. 2008, Wei et al. 2019). GmZF351, a member of the CCC zinc finger protein family, promotes leaf stomatal closure by activating the expression of GmSnRK, thereby improving soybean drought resistance (Wei et al. 2023). Through the genome-wide association study (GWAS) of soybean field drought resistance index, an oxidase GmPrx16 (Peroxidase) that responds to soybean drought stress was identified. Overexpression of GmPrx16 significantly increased the peroxidase activity of soybean leaves, reduced the content of reactive oxygen, and thus improved the drought resistance of soybean (Zhang et al. 2024). The above research mainly focuses on the mechanism analysis of abscisic acid-mediated drought stress response.

[0005] Ent-kaurenoic acid oxidase (KAO), a key enzyme in the biosynthesis of gibberellic acid (GA), is a cytochrome P450 monooxygenase of the subfamily CYP88A that catalyzes the conversion of ent-kaurenoic acid (KA) to GA. 12 The conversion of KAO provides precursors for the synthesis of biologically active GAs. Recent studies have shown that KAO plays an important role in the growth and development of soybeans. There are two KAO genes in Arabidopsis, which are highly expressed in developing organs such as germinating seeds and inflorescences. The functional loss of the Arabidopsis KAO gene reduces the active content of GA in the plant, resulting in a typical dwarf phenotype (Regnault et al. 2014). ZmGAD5 encodes a KAO protein. The functional loss of ZmGAD5 significantly increases the KAO content and greatly reduces the GA content. 12 and active GA3 content, ultimately leading to plant dwarfing and a significant shortening of internode length, but without changing the root development and internode number of the plant (Li et al. 2023).

[0006] However, whether soybean KAO is involved in the regulation of soybean drought stress needs further exploration. Summary of the invention

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

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

[0009] The first aspect of the present invention provides the use of soybean GmKAO gene in improving soybean drought resistance, characterized in that the improvement of soybean drought resistance is achieved by any of the following methods:

[0010] (1) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously;

[0011] (2) causing the deletion of 118 bp to 122 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2;

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

[0013] The second aspect of the present invention provides the use of soybean GmKAO gene in breeding drought-resistant soybean varieties.

[0014] The third aspect of the present invention provides the use of a knockout vector of the soybean GmKAO gene in improving the drought resistance of soybean or cultivating drought-resistant soybean varieties.

[0015] The fourth aspect of the present invention provides the use of an engineered bacterium transformed with a knockout vector of the soybean GmKAO gene in improving the drought resistance of soybean or cultivating drought-resistant soybean varieties.

[0016] The fifth aspect of the present invention provides a method for improving drought resistance of soybean, which comprises the following steps: causing the 120bp~124bp of the GmKAO1 gene shown in SEQ ID NO:1 and the 105bp~110bp of the GmKAO2 gene shown in SEQ ID NO:2 of the soybean plant to be deleted at the same time, the 118bp~122bp of the GmKAO1 gene shown in SEQ ID NO:1 and the 105bp~110bp of the GmKAO2 gene shown in SEQ ID NO:2 to be deleted at the same time, or the 120bp~124bp of the GmKAO1 gene shown in SEQ ID NO:1 and the 109bp~112bp of the GmKAO2 gene shown in SEQ ID NO:2 to be deleted at the same time.

[0017] The inventors of the present invention found that by knocking out the GmKAO1 gene and GmKAO2 gene of soybean plants at the same time, the GmKAO1 gene and GmKAO2 gene were partially deleted, and the survival rate of the mutant strain, the soil water content during drought stress, the relative water content of leaves, and the proline content were significantly higher than those of the wild plant TL1; the anthocyanin, propylene glycol MDA content, and H2O2 content were significantly lower than those of the wild plant TL1, indicating that the GmKAO gene negatively regulates the drought resistance of soybeans. The discovery of the new function of the soybean GmKAO gene provides new gene targets and resources for drought-resistant soybean genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0019] Figure 2 The target site sequencing results of the soybean GmKAO gene knockout mutant strain in Example 2 of the present invention, wherein TL1 is a wild-type soybean, and gmkao-1, gmkao-2 and gmkao-3 are GmKAO gene knockout mutants.

[0020] Figure 3 These are the results of drought tolerance identification of soybean GmKAO gene knockout strains in Example 3 of the present invention, wherein TL1 is 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 invention, the present invention will be described more fully below. The present invention can be implemented 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 understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0023] Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2013), or the conditions recommended by the manufacturer's instructions.

[0024] In some embodiments of the present invention, 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 methods:

[0025] (1) Deleting 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2;

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

[0027] (3) Deletion of 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and deletion of 109 bp to 112 bp of the GmKAO2 gene as shown in SEQ ID NO: 2.

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

[0029] In other embodiments of the present invention, the use of a knockout vector of the soybean GmKAO gene in improving the drought resistance of soybean is disclosed, wherein the improvement of the drought resistance of soybean is achieved by any of the following methods:

[0030] (1) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously;

[0031] (2) causing the deletion of 118 bp to 122 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2;

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

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

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

[0035] S1. Using soybean leaf DNA as a template and SEQ ID NO: 5 and SEQ ID NO: 6 as primers, amplify the GmU6 promoter fragment;

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

[0037] 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, amplify to obtain the GmU6-sgRNA fragment;

[0038] S4. Connect the GmU6-sgRNA fragment and the CRISPR vector after enzyme digestion.

[0039] In other embodiments of the present invention, the use of a knockout vector of the soybean GmKAO gene in breeding drought-resistant soybean varieties is disclosed.

[0040] In other embodiments of the present invention, disclosed is the use of an engineered bacterium transformed with a knockout vector of the soybean GmKAO gene in improving the drought resistance of soybean.

[0041] In other embodiments of the present invention, disclosed is the use of an engineered bacterium transformed with a knockout vector of the soybean GmKAO gene in breeding drought-resistant soybean varieties.

[0042] In other embodiments of the present invention, a method for improving drought resistance of soybean is disclosed, which comprises 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 of the soybean plant, 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.

[0043] In some embodiments, the deletion is achieved by the following steps: after infecting soybean cotyledon nodes with engineered bacteria transformed with a knockout vector for the soybean GmKAO gene, soybean tissue culture is performed to obtain tissue culture seedlings, and then a transgenic strain (i.e., a soybean GmKAO gene knockout strain) is obtained.

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Example 1 Construction of soybean GmKAO gene knockout vector

[0046] In this example, the Infusion system (Novozyme) was used to construct the GmU6 promoter and the sgRNA containing the 19bp target site sequence (TGGCTCTATGAATCCAAAT) into the CRISPR vector through the restriction enzyme cutting site XbaI, and a vector (named CRISPR-GmKAO12) for simultaneously knocking out the soybean GmKAO1 gene and GmKAO2 gene was obtained, wherein the CDS sequence of the soybean GmKAO1 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO: 3, and the CDS sequence of the soybean GmKAO2 gene is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by it 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] Amino acid sequence of mKAO2 (SEQ ID NO:4): ATGGACAATTGCTTGGGAAGGGTCAGGAAATGTTCATCTACAACAACCTAAG

[0114] MMEMDSMCMWVVLVAIAGALLVLRSMLKNVNWWLYESKLGVKQYSLPPGDM

[0115] GWPFIGNMWSFLRAFKSKDPDSFISSFVSRYGRTGMYKTLMFGNPSVIVTTPETCK

[0116] RVLTDDDKFTTGWPQSTIELIGKRSFISMSYEEHKRLRRLTSSSINGMESLSLYLTYI

[0117] EENVKNSLEKWANMGQIEFLTEIRKLTFKIIMHIFLSSESEPVMEALEREYTALNHG

[0118] VRAMCINIPGFAYHKAFKARKNLVAIFQSIVDERRNLRKGYLPGKAKDMMDALID

[0119] VEDDDGRKLSDEDIIDIMLMYLNAGHESSGHITMWATFFLQKHPEYLQKAKAEQE

[0120] EIIRRRPPTQKGLTLKEVREMDFLYKVIDETLRVITFSLVVFREAKSDVNINGYTIPK

[0121] GWKALVWFRSVHLDPEIYPNPKEFNPYRWNKEHKAGEFLPFGGGSRLCPGNDLAKMEIAVFLHHFLLNYRFEQHNPNCPVRYLPHTRPMDNCLGRVRKCSSTTT*

[0122] Method for constructing gene knockout vector, specifically comprising the following steps:

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

[0124] (1) Take an appropriate amount of young soybean leaves and place them in a 1.5 mL RNase-free centrifuge tube filled with proofing steel beads. Place the tube in liquid nitrogen immediately. Grind the tube in a frozen state using a proofing machine or a mortar. Add 500 μL of 65°C preheated CTAB solution, vortex to mix, and place the tube in a 65°C drying oven for 30 min.

[0125] (2) Remove the sample from the drying oven and cool it. Add 500 μL of 25:24:1 Tris-saturated phenol / chloroform / isoamyl alcohol solution (the solution must be cooled to room temperature before adding), mix by inverting or vortexing, centrifuge at 12,000 rpm for 15 min at room temperature, and aspirate 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 by inversion or vortexing. Centrifuge at 12,000 rpm for 10 min at room temperature. Slowly pipette 500 μL of supernatant into a new centrifuge tube.

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

[0128] (5) Discard the supernatant, add 1 mL of 75% ethanol, mix by inversion, and centrifuge at 12,000 rpm for 10 min at room temperature. Discard the supernatant again, let it stand at room temperature for 5 min, remove the excess ethanol with a pipette, let it stand at room temperature for 20 min, and add 40 μL of ultrapure water to dissolve it for later use.

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

[0130] SEQ ID NO:5:

[0131] GGAAGCTTAGGCCTTCTAGAAAAATAAATGGTAAAATGTC

[0132] SEQ ID NO:6:CAATCCATGTGGTGGCACAT

[0133] Using the sequence shown in SEQ ID NO: 7 as a template, Gmkao-F (SEQ ID NO: 8, wherein the underline is the target site sequence of Gmkao1 and Gmkao2 genes) and sgRNA-XbaI-R (SEQ ID NO: 9) as primers, the sgRNA fragment (TGGCTCTATGAATCCAAAT, SEQ ID NO: 10) containing the target site (19 bp) sequence of Gmkao1 and Gmkao2 genes was amplified, and the PCR amplification reaction system and reaction procedure are shown in Tables 1 and 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: GCAACGCGTTCTAGAAAAAAAAAGCACCGACTCGGTGCCAC

[0141] Table 1

[0142]

[0143] Table 2

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

[0145] 3. Using the GmU6 promoter fragment obtained in step 2 and the sgRNA fragment containing the 19bp target site sequence as templates, Gmkao-F (SEQ ID NO: 8) and sgRNA-XbaI-R (SEQ ID NO: 9) as primers, amplify the GmU6-SgRNA fragment containing the Gmkao1 and Gmkao2 gene target site (19bp) sequences. The PCR amplification reaction system and reaction procedure are shown in Table 1 (except for the different templates, which are input at 1:1, everything else is the same) and Table 2.

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

[0147] Table 3

[0148]

[0149] 5. Perform gel electrophoresis on the PCR product of step 3 and the vector after enzyme digestion in step 4 to determine the size of the target band and remove it (use Axygen gel recovery kit to recover various corresponding products, and follow the instructions of Axygen for detailed experimental steps). Place the target band in a 1.5mL centrifuge tube and connect at 37℃ for 30min (the connection reaction is shown in Table 4) to obtain a CRISPR vector with simultaneous knockout of soybean GmKAO1 gene and GmKAO2 gene (named CRISPR-GmKAO12, with a structure as shown in Figure 1 Store at 4°C.

[0150] Table 4

[0151]

[0152] Example 2 Obtaining soybean GmKAO gene knockout plants

[0153] In this example, a genetic transformation method mediated by Agrobacterium EHA105 was used to transfer the CRISPR vector for simultaneously knocking out the soybean GmKAO1 gene and GmKAO2 gene constructed in Example 1 into the soybean cotyledon node to obtain a soybean plant with simultaneously knocking out the soybean GmKAO1 gene and GmKAO2 gene, which comprises the following steps:

[0154] 1. Preparation of competent Agrobacterium

[0155] Pick a single colony of Agrobacterium tumefaciens EHA105 and place it in 5 mL of LB liquid medium containing the corresponding antibiotics. The resistance of EHA105 is: 100 μg / mL rifampicin (Rif). Culture at 28°C overnight; take 500 μL of the overnight culture solution and inoculate it in 50 mL of LB liquid medium containing the corresponding antibiotics, and culture at 28°C until the OD 600 About 0.5; place on ice for 30 minutes; centrifuge at 4°C, 5,000rpm for 10 minutes, resuspend the Agrobacterium cells with 15mL pre-cooled 10mM CaCl2, centrifuge at 4°C, 5,000rpm for 10 minutes; resuspend the precipitate with 2mL pre-cooled 10mM CaCl2, dispense into 100μL / tube on ice, quick-freeze with liquid nitrogen, and store at -80°C.

[0156] 2. Electroporation of Agrobacterium

[0157] Add 3 μL of plasmid (i.e., the soybean GmKAO gene knockout vector constructed in Example 1) to 50 μL of Agrobacterium competent state, mix by aspirating with a sterilized gun tip, and let stand on ice for 30 minutes. During this period, prepare an electroporation cup and clean bench to dry and pre-cool. Transfer the competent state to the electroporation cup on ice and prepare for electroporation transformation. Use BIO-RAD Transformer (Gene Pulser Xcell Electroporation System) Bacterial electroporation transformation. After electroporation, add 700 μL of antibiotic-free LB liquid culture medium to the clean bench to resuscitate the bacterial solution, shake at 28°C and 200rpm to resuscitate the bacterial solution for 2 hours, collect the bacterial solution by centrifugation and apply the plate. Cultivate for about 2-3 days, wait for Agrobacterium to grow up, and screen Agrobacterium monoclones according to the method for screening positive Escherichia coli monoclones.

[0158] 3. Agrobacterium infection of soybean cotyledon nodes

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

[0160] (1) The positive Agrobacterium single clone screened in step 2 was incubated in a 10 mL test tube and shaken overnight.

[0161] (2) Sterilize soybeans in a sealed container with 100 mL of Kao bleach + 4 mL of concentrated hydrochloric acid for 16-18 hours. Place the soybeans in a sterilized clean bench for 30 minutes to blow away the chlorine.

[0162] (3) Prepare co-cultivation solid / liquid culture medium, water, filter paper and conical flask, and sterilize at 121℃ for 20 minutes. Soak beans in sterilized water for about 8 hours (generally, place beans in a dark incubator at 28℃ between 6 and 10 pm and soak for about 8 hours. Beans should not be soaked for too long). At the same time, shake 50 mL of Agrobacterium in a conical flask for infection.

[0163] (4) Take the soaked beans and use a surgical blade to cut the soybeans in half, into two bean pieces. Cut off the excess hypocotyl 3mm to 4mm from the hypocotyl. Use a blade to gently cut the hypocotyl cotyledon node 5 to 7 times, 0.5mm deep. After cutting, place the bean pieces in a conical flask with water.

[0164] (5) Wait until the OD of Agrobacterium 600 =0.5-0.7, centrifuge at 4,000 rpm for 10 minutes, collect the bacterial solution, remove the supernatant, add an appropriate amount of co-culture liquid medium, vortex and mix until the bacterial solution OD 600 =0.6.

[0165] (6) After cutting the beans, remove the water in the conical flask, add the bacterial solution prepared with the co-cultivation liquid culture medium, shake 2 to 3 times, and infect for 30 minutes. Shake it several times in between. At the same time, spread the sterilized filter paper on the co-cultivation solid culture medium, with one filter paper corresponding to one dish of culture medium. After the infection is completed, remove the bacterial solution, place the infected beans on an empty culture dish containing several layers of filter paper, absorb the excess bacterial solution, and then evenly place the beans on the co-cultivation solid culture medium covered with filter paper. Incubate in the dark at 28℃ for 3 days. 20 to 30 beans can be spread on one dish of co-cultivation solid culture medium. It is not advisable to spread too many beans on one dish of co-cultivation solid culture medium to prevent poor growth of the beans and cross contamination.

[0166] (7) Prepare induction solid / liquid culture medium, water, filter paper and conical flask, and sterilize at 121℃ for 20 minutes. Remove the bean sprouts and excess hypocotyls that have been co-cultured for three days, leaving 3mm to 4mm of hypocotyls, and place them in a conical flask with water. Wash with sterile water 4 to 5 times, wash with induction liquid culture medium 4 to 5 times, place on an empty culture dish containing filter paper, and absorb the water. Then insert the bean sprout hypocotyls obliquely upward into the induction solid culture medium and culture under light for 10 days.

[0167] (8) After 10 days, remove the larger embryo at the base of the bean, gently cut 5 to 7 places on the hypocotyl, insert the bean with the hypocotyl facing upward obliquely into the new induction solid medium, and culture under light for 10 days. Repeat twice.

[0168] (9) Prepare elongation solid culture medium and sterilize at 121℃ for 20min. After three induction cultures, most bean petals grow callus with embryos. Cut off the bean petals, keep the callus, remove the black tissue on the surface of the callus, place the remaining callus in elongation medium, and culture under light for 10 days. Repeat three times.

[0169] (10) During the elongation medium period, when the callus embryo elongated to a length greater than 3 cm, it was cut from the callus and placed in a rooting medium. It was cultured under light for 10 to 14 days. After the seedlings grew roots, they were transferred to soil and hardened for about 7 days to obtain T0 generation transgenic seedlings.

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

[0171] Leaves of T0 and T1 transgenic seedlings were taken to extract DNA, and PCR amplification was performed using primer pairs GmKAO1-F (SEQ ID NO: 11) and GmKAO1-R (SEQ ID NO: 12), GmKAO2-F (SEQ ID NO: 13) and GmKAO2-R (SEQ ID NO: 14), respectively, with a reaction volume of 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 was completed, 5 μL of the sample was taken for gel running to detect whether there were specific bands, and the remaining 45 μL of the sample was sent to Qingke Biotechnology Co., Ltd. for first-generation sequencing and compared with the wild-type genomic DNA to determine whether the soybean GmKAO gene knockout strain was a homozygous mutation. Finally, three homozygous mutant soybean GmKAO gene knockout strains (mutants) were obtained, named gmkao-1, gmkao-2 and gmkao-3, respectively. The target sequencing results of the gene knockout strains are shown in the figure. Figure 2 As shown, from Figure 2 It can be seen that in the mutant gmkao-1, the GmKAO1 gene deleted 5bp and the GmKAO2 gene deleted 6bp; in the mutant gmkao-2, the GmKAO1 gene deleted 5bp and the GmKAO2 gene deleted 6bp; in the mutant gmkao-3, the GmKAO1 gene deleted 5bp and the GmKAO2 gene deleted 4bp.

[0177] Example 3 Identification of Drought Tolerance of Soybean GmKAO Gene Knockout Lines

[0178] Tianlong No. 1 (control TL) and soybean GmKAO gene knockout lines 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 small pots and subjected to drought stress by withholding water for 2 weeks. Water supply was then restored. The traits of Tianlong No. 1 and the gene knockout lines gmkao-1 and gmkao-3 were observed before water withholding (control), 6 days after water withholding, and 10 days after water restoration. Figure 3 A) in the figure, and conduct drought tolerance test 10 days after water supply is restored.

[0179] The results showed that the soil water content of gmkao-1 and gmkao-3 plants during drought stress ( Figure 3 B) leaf relative water content ( Figure 3 C), survival rate ( Figure 3 D), proline content ( Figure 3The anthocyanins accumulated in gmkao-1 and gmkao-3 plants under drought stress ( Figure 3 E), MDA content ( Figure 3 G) and H2O2 content ( Figure 3 The H) in TL1 is significantly lower than that in TL1.

[0180] The results of this example show that by simultaneously knocking out the gibberellin synthesis genes GmKAO1 and GmKAO2 and terminating them early, the drought resistance of soybean can be improved, proving that GmKAO1 and GmKAO2 negatively regulate the drought resistance of soybean, and the drought resistance of gmkao-3 plants is significantly stronger than that of gmkao-1 plants.

[0181] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0182] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of soybean GmKAO gene in improving drought resistance of soybean, characterized in that: The improved drought resistance of soybean is achieved by any of the following methods: (1) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously; (2) causing the deletion of 118 bp to 122 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2; (3) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 109 bp to 112 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously.

2. Application of soybean GmKAO gene in breeding drought-resistant soybean varieties, characterized in that: The cultivation of drought-resistant soybean varieties is achieved by any of the following methods: (1) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously; (2) causing the deletion of 118 bp to 122 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2; (3) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 109 bp to 112 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously.

3. Application of a knockout vector of soybean GmKAO gene in improving drought resistance of soybean, characterized in that: The improved drought resistance of soybean is achieved by any of the following methods: (1) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously; (2) causing the deletion of 118 bp to 122 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 105 bp to 110 bp of the GmKAO2 gene as shown in SEQ ID NO: 2; (3) 120 bp to 124 bp of the GmKAO1 gene as shown in SEQ ID NO: 1 and 109 bp to 112 bp of the GmKAO2 gene as shown in SEQ ID NO: 2 are deleted simultaneously.

4. The use 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 use according to claim 4, characterized in that: The knockout vector of the soybean GmKAO gene is constructed by the following method: S1. Using soybean leaf DNA as a template and SEQ ID NO: 5 and SEQ ID NO: 6 as primers, amplify the GmU6 promoter fragment; S2. Using SEQ ID NO: 7 as a template and SEQ ID NO: 8 and SEQ ID NO: 9 as primers, amplify a 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, amplify to obtain the GmU6-sgRNA fragment; S4. Connect the GmU6-sgRNA fragment and the CRISPR vector after enzyme digestion.

6. Application of the knockout vector of soybean GmKAO gene in breeding drought-resistant soybean varieties.

7. Application of engineered bacteria transformed with a knockout vector of the soybean GmKAO gene in improving soybean drought resistance.

8. Application of engineered bacteria transformed with a knockout vector of the soybean GmKAO gene in breeding drought-resistant soybean varieties.

9. A method for improving drought resistance of soybean, characterized in that: It includes the following steps: The soybean plant is caused to simultaneously delete 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, 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 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.

10. The method for improving drought resistance of soybean according to claim 9, characterized in that: The deletion is achieved by the following steps: after the engineered bacteria transformed with the knockout vector of soybean GmKAO gene are infected with soybean cotyledon nodes, soybean tissue culture is performed to obtain tissue culture seedlings, and then a transgenic strain is obtained.

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