CN115960192A - Application of GmASHH2a/b protein and coding gene thereof in regulating and controlling late flowering of soybean - Google Patents
Application of GmASHH2a/b protein and coding gene thereof in regulating and controlling late flowering of soybean Download PDFInfo
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Abstract
The invention belongs to the technical field of biotechnology, and particularly relates to an ASHH2a/b protein and application of a coding gene thereof in regulating and controlling soybean late blossoming. The invention designs 2 sgRNA for editing genes aiming at conserved segments of 2 soybean ASHH2s genes, constructs a set of CRISPR-Cas9 vectors, and performs agrobacterium-mediated genetic transformation to obtain soybean late flower mutants. Therefore, the invention provides the application of the ASHH2s gene in the creation of soybean late-flowering mutants. Meanwhile, the invention also provides application of the ASHH2s gene knockout vector in creating soybean late-flowering mutants. The ASHH2 gene is determined as a target gene for creating the soybean late-flowering character, and a soybean late-flowering mutant is successfully created, so that a material basis is provided for the follow-up research of soybean flowering phase regulation and late-flowering related breeding and screening.
Description
Technical Field
The invention belongs to the technical field of biotechnology, and particularly relates to application of GmASHH2a/b protein and a coding gene thereof in regulation and control of soybean late blossoming.
Background
Soybean (Glycine max), an annual herbaceous plant, originated in china, is an important food and oil crop and also a high-quality vegetable protein food source. The soybean is rich in nutrient components, high in protein content, and contains amino acids, fat and various mineral nutrient elements which are necessary for human bodies.
The soybean yield is determined by the agronomic characters such as flowering phase, mature phase, plant height, main stem node number, effective branch number, pod bearing habit and the like. Soybeans are typically short day plants and are extremely photoperiod sensitive. The flowering time determines the regional adaptability, the sowing time and the harvesting time of the soybeans. When flowering at a proper time, the conversion from vegetative growth to reproductive growth is an important node for the completion of the life cycle of soybeans, and is very important for improving the yield of the soybeans. The Chinese invention patent application No. 202110555960.4 discloses the application of GmEID1 protein in regulating soybean flowering and main stem node number. Chinese patent application No. 202110086368.4 discloses a protein related to plant flowering phase and its coding gene and application. A functional study on the regulation of the flowering phase and plant height by the GmGAMBB gene discloses the regulation of the flowering phase and plant height of soybeans by the GmGAMBB gene of soybeans. Therefore, the method has important theoretical significance and application value for excavating yield potential by excavating related functional genes for controlling flowering phase, regulating plant height and the like in soybeans and clarifying molecular mechanisms of the genes.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides the following technical solutions:
the application of the GmASHH2a/b protein in regulating and controlling the growth and development of plants is at least one of the following:
(1) The GmASHH2a/b protein is applied to controlling the flowering phase of plants;
(2) The GmASHH2a/b protein is applied to increasing the effective pod number of a plant single plant;
(3) The GmASHH2a/b protein is applied to increasing the number of main stem nodes of plants;
(4) The GmASHH2a/b protein is applied to increasing effective branches of plants;
the amino acid sequence of the GmASHH2a protein is shown as SEQ ID NO. 5; the amino acid sequence of the GmASHH2b protein is shown as SEQ ID NO. 7. The nucleotide sequence of the GmASHH2a gene for coding the GmASHH2a protein is shown as SEQ ID NO. 4; the nucleotide sequence of the GmASHH2b gene for coding the GmASHH2b protein is shown as SEQ ID NO. 6.
Preferably, the plant is soybean.
Preferably, the controlling plant flowering is delayed soybean flowering.
A method of growing soybeans, the method comprising the steps of:
(1) Constructing a GmASHH2a/b gene knockout vector;
(2) Carrying out soybean genetic transformation;
(3) Screening and identifying transgenic materials.
Preferably, the GmASHH2a/b gene knockout vector comprises sgRNA1 and sgRNA2; the sgRNA1 is shown as SEQ ID NO:8 is shown in the specification; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO: shown at 9.
Preferably, the vector is pCBSG015.
The invention has the beneficial effects that:
the invention designs 2 sgRNA for editing genes aiming at conserved segments of 2 soybean ASHH2s genes, constructs a set of CRISPR-Cas9 vectors, and performs agrobacterium-mediated genetic transformation to obtain soybean late flower mutants. Therefore, the invention provides the application of the ASHH2s gene in the creation of soybean late-flowering mutant. Meanwhile, the invention also provides application of the ASHH2s gene knockout vector in creating soybean late-flowering mutants. The invention determines the ASHH2 gene as a target gene for creating the soybean late-flowering character, successfully creates a soybean late-flowering mutant, and provides a material basis for the subsequent research of soybean flowering phase regulation and late-flowering related breeding and screening.
Drawings
FIG. 1 is a structural diagram of a GmASHH2a/b gene knockout vector construction.
Figure 2 is a PCR detection map of the Cas9 gene.
FIG. 3 is a PCR detection map of GmActin gene.
FIG. 4 is a PCR detection map of the GmASHH2a gene.
FIG. 5 is a PCR detection map of the GmASHH2b gene.
Fig. 6 is a schematic diagram of CRISPR/Cas9 gene editing target sites.
FIG. 7 is a scheme of genetic transformation of soybean.
FIG. 8 is field phenotype and mutation type analysis of T1 generation CRISPR/Cas9 gene editing plants.
Fig. 9 is CRISPR/Cas9 gene editing plant mutation type analysis.
Figure 10 is a plant late flower phenotype edited by CRISPR/Cas9 gene.
Fig. 11 is a field late flowering phenotype of CRISPR/Cas9 gene edited plants.
Fig. 12 shows plant type and seed characteristics of CRISPR/Cas9 gene editing plants at maturity.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Test examples
1 method
1.1GmASHH2a/b Gene cloning
In the soybean database: (https://phytozome-next.jgi.doe.gov/) The gene sequences of GmASHH2a (Glyma.06G117700) and GmASHH2b (Glyma.04G24540) are downloaded, and primers are designed as follows:
GmASHH2a F: ATGGTTGAGATGGGGGTCGTG (shown in SEQ ID NO: 1);
GmASHH 2R: TTACTCCTCTCTACAGATTGCATCCC (SEQ ID NO: 2);
GmASHH2b F: ATGATTGAAATGGGGTCGTGTG (shown in SEQ ID NO: 3);
nuozantin high fidelity enzymatic amplification system: 2 × Phanta-Master Mix 25 μ L;18 μ L ddH 2 O; 2.5. Mu.L of forward primer; 2.5. Mu.L of reverse primer; mu.L of cDNA template. The PCR reaction conditions were as follows: pre-denaturation at 98 ℃ for 5min; 30sec at 98 ℃, 30sec at 58 ℃, 4min at 72 ℃ and 30 cycles; extension at 72 ℃ for 5min. After DNA purification and recovery, the ligation cloning vector was sequenced. 4 mu L of DNA purified product; 1 μ L of pEASY-Blunt T vector was placed in a water bath at 24 ℃ for 25min. mu.L of the ligation reaction system was added to the initially-thawed E.coli competent cells Trans1-T1, incubated in an ice bath for 5min, heat-shocked in a water bath at 42 ℃ for 1min, and placed back on ice. Adding 1mL of LB liquid culture medium precooled at 4 ℃, performing shaking culture at 37 ℃ for 1h, sucking 200 mu L of bacterial liquid, coating the bacterial liquid on an LB + Kan plate, and placing the plate in an incubator at 37 ℃ for culture for 16h. Single colonies were picked and subjected to colony PCR. And (3) PCR system: 2 × Rapid Taq Master Mix 7.5 μ L;5.5 μ LddH 2 O; the forward primer (35s F. The reaction conditions for PCR amplification are as follows: pre-denaturation at 98 ℃ for 5min; 25sec at 98 ℃, 25sec at 58 ℃, 25sec at 72 ℃,30 cycles; extension at 72 ℃ for 5min. Picking out positive single colony, carrying out shake culture on 10mL LB + Kan liquid culture medium at 37 ℃ for 16h, and carrying out sequencing on a bacteria liquid sample.
(1) GmASHH2a nucleotide sequence: (6123 bp) SEQ ID NO:4 is shown in
ATGGTTGAGATGGGGTCGTGTGGGAGATCTGCTGCCATTGATAATCCCTCAGAAAAGTTTATTATTGAGCAGCAGCAGCATTTGTGCTTAGAGGTTCAAGAGCAGGAGGTTGTTTCTGTTGTGCAGGAGTCTTGTTTAGAGGAAGAGGCTTGCAATGTGGTGGACTCTAATGTTGAGCTCTCCACTGTGATTGATGGTTGTCTGGGAGAGGACCGTGTCTGTTCCAAACGATGTGTTGATGTTACCGAGGGGTCCAGAGAGGGTTTGGGTTTGGGTTTGGTGAGTGAATGCGAAAATGCTGATTTGTTGCCACTGGAGAAATCTGCCGAAGATGATTGCCAGAATTATTTGGGGGTTTCTTGTGGGAGCATTGAAGTTCCCTGTGTAAACAGTGGCTCAGAGGGAAGATTTCTGGATGAGGGTAACTTTGATCTGCCTTCGGGGTCCTTAACTGCAGATGATTCACAGATGTATTGTGCTCAACTGGATGAGCAGAAGGATGATGAGTCATGGTCCTTAATTACTGATGATTCACAGAGGCATTGTGCTCAACAGGATGAGCAGAAGGATGATAAGAGCGATGTATTGCCTGCAGCAGGAGATGATTTGGCTGTCGTGGAAGGCAAAAACGATGTCACTGGTGTTCTTGCTGATGCTTTCAGTCATGTACATGATTTCAGGGATTGCGAAGTGTCTTTGGAATCGGAATCCATGGCTGACTTGTTAGTTGATTGCAACTGGCAAAGTGAGCAGGAGGAAATTATGAGAAATACTGATCCCTTGTTGAATGTAGTGGAAAAATGTGACGCTCTAATTGTGGAGGAAACTGATGCTTGCAGGCAGATATCTCCTACACCGGCTATGGAAGTGCCATCAGGGGCACCATCAGGTGCATTATGTACAGATACTGAAGTAGAGAGCACAAGTGATCAGCCATGTGATCAGAAAGATGGTGAGGAAACTGATGTTTGCAGGCAGATATCTCCTACTCTGGCTATGGAAGTGCCATCAGGGGCATTTTGGACAGATACAGAAGTAGAGAGCACAAGTGATCAGCCATGTGATCCGAAAGATGGTGAGGATCAAAATAGTACTTGTGAAGAAATCATCAACATTAATTCATGTGTAAAAATATCATCCTCTCCAGGTTGCAATGAGACTGTTGGGAGCTCACCTGTAGTTGGTTTTCCTTGTGAGCCTGCCCTGTTGGATCCAGAGTATGAAATGAAGAATGGCATGCTGCAAATAGAAGATGATGCCTGTAAATTGAAGGACTGTTCCTCAGAGGAAACTACCAACTCTACCTTTAGAAAACCATTTTCTCCAGAATCAGGTCTGCCCTCTGTTGCTTTAATTACCAACTGCTCTGCAAAGGATGTTCCTGATCAACAGTCTAAGGGTGATGATGTTTCCATTGATAATAATAATGCTGTGAACAATCAAGGGCAGATGGATAATGATGGAACAGAAGCTGTTGAAGTTGATTGTATCACTGAAGGCATACCGTTACCTTCTCAAAGGAATAGTCGAAGAACTAAATTTGGCCGTAAGACACAAACCAAAAAGGCTTCAAGAAAATGCAAGAACAAAACCAAGGTTACACATCCAAATGGGGGTATGAAGCTAAACTTAGAGGCTGCTAGAAAGAAAAGAAGTTGTTTCTCCAAACCAGCTCGTTCTTCTATCTGGGGATTGATTGGAAATATTGAACAGTTTTTTGAGCAAGATAATGAGCTTGGAGATGGTGAAGCTGTGTGCCAAGAATTGGGGAAGGCTAGAAGCAAGCCTCAAAGTGGGAAAGCGGTTAAGAATGGTGCAAGTACTACCTCATTGGGTTCAGTACAGAAGCATTCTGTTTCAACTACTCGTGTTCGTCTGAAGATCAAATTTGGGAAAGAAGTTGATTTAAGTTGCTCAAATGTCTTGATTCCAGAGTCTGTTGATGGCTTGGCTTCTGCTTCCTATTTGGGGTCTGGTTCAGGTTCTCAGAAAGTAGCTGGCAATGCTGATGATAAAATTTCTGAAGTGGTGGCTTTGGGCCATTCAGAATCTTTCAATAATGACCTGGACAAGGATGGTTTTGTTCTAAATGAACAAGTTGCAAATAACCCCTTAGAAACCACTGAAATAACAGAGAAGTCATATGGGGATGCAGAGGAACCTTGTCTTGCAGTTCCTCCAGAGAAGGTGGTTGAAGCATTGATTGAACCTATCAATAACAAGGGTATGGATCCTGGAACCTCACCTGATTCTGAAGTTATCAATTCAATTCCCGAAGTCCAGGCTGGAGAAAAACATCAAGAAGATGCACATCATGCTGTTTTAGGTTCTTCTAAAGAATTAAATTCTAAATTGGATGTTACCATCAGTAAGAGAGGGAAGAACAAAGAGAAAGTTATTTGTTCAAGTAATTGTATCACTGAAGATGGATCACAAGGTCCACATAAGAATAGTAGAGCTAAGCATTCAAAGAATCACAGACGTAAGAAAAATTGCAGGGATGTGGTCAGCTCTTTGGAATTGCCCACTGATATAAGCAAATCTTTGAGCAGTAAAGAATTGTCCCCAGAATCATTGCCTCTTTCTGTAGAGACTGAACTTGGAGGCTCCACTGAGGCTTTGAAAGTTAAAAATCATACGGACGTTAAGACAAGTGACAAACCATCTGTTGACCATGGATTTTCAGATTCCCTGGTTGCTGAGAATATGCTGTCATCTGCAAGACCTTTGGAGCGTAAACTACCTAAAAGTCTTAGAGCTAGTAAAGTTAGCAAGACCAAATCTAAAGCTTCTGACTCAACTGGCAGGAAAAAGACTACTGCTGGTATACGCAAGGAGAAACAGATAAAGGCAATTAATAAGAGCAAAGTCAAGGGAAAAGGTGTTTCTCTTAAAGTTACATGTGAAGTGGAAGATTGCCTGCATCCAGAAGAAAATGCTGGAAATCATAAGCTGGATGCTGTTGGAAAAATTATTGCTGATGACAACAGAGTATCGGTCAATTTATCTAATTTGGACATGCTGTCTGGTGTTGGTTATGGGGAGCAACTTCTATCACCCCGTAATGCATGGGTTCGTTGTGATGATTGTCACAAGTGGCGGCGAATTCCAGCTGTGCTTGCAGACCGAATTGATGAAACTAACTGCACCTGGACATGTAAGGACAGCAGTGATAAAGCCTTTGCTGATTGTGCTATCCCTCAAGAAAAGTCAAATGCAGAGATTAATGCGGAGTTGGGATTATCAGATGCCTCGGGTGAAGAAGATGCATATGAAGGTTCCAAAAATTTTAAGGAATTAGAATATCGGCCTCCGTTAGTTTCCCAGGAGTCAACTTTTACCCATATTTTGACCAATGAATTTCTGCATCGTAGCCATAAAACTCAGACTATTGATGAGATAATGGTCTGTCATTGCAAGCCATCTCAAGAAGGAAAGTTGGGCTGTGGGGATGAATGTCTAAATCGGATTCTTAATATTGAATGTGTGCAAGGAACCTGCCCATGCGGGGACCGTTGTTCCAACCAGCAGTTCCAAAAACACAAGTATGCTAGCCTGAAGTGGTTTAAATGTGGGAAAAAGGGTTATGGACTGAAGGCAATTGAGAATGTAGCTCAAGGCCAGTTTCTTATTGAATACGTTGGAGAGGTACTTGATATGCAAGCATACGAGGCACGGCAAAGAGAGTATGCTTTGAAGGGTCATCGGCATTTCTATTTTATGACCTTGAATGGCAGTGAGGTGATAGATGCAAGTGCAAAAGGAAACTTGGGGCGTTTCATTAATCATAGTTGTGATCCTAATTGTCGGACAGAAAAGTGGATGGTGAATGGGGAAATCTGTATTGGACTGTTTGCATTGAGGGATATTAAGAAGGATGAAGAATTGACCTTCGATTACAACTATGTGAGGGTTTTTGGTGCTGCTGCCAAAAAATGTTATTGTGGTTCACCTAACTGTCGAGGTTATATAGGTGGTGGTGATCCACTTAATGCTGAACTGATAGTTCAAAGTGATTCAGAAGAAGAATTTCCAGAACCTGTCATGCTCACCAAAGATGGTGAAATTGAAGATTCTGTACCTACACCCGAGTATTTTAATAATGTTGATACACAATCTGCTAAACATATGTTGAAAGACAGGGATATATTGGACAATTCTACAACTGCTATAGATTCGGATGGTTCTCTAGAGAAAGAGCGTTCTATGAACCCTGCCTCTGCTGTTTCTCTGTTGCACAGCTCAGCAGAAATGGAGGATTCAAAGGGTAAATTACAATCTTCTGTTCAAGTTGAAGAAATTTCTCAACAAATGGAGGATGTAACAAGCAAACCCATGCCTGCTGTACACCAAGGATATGAAAAGGAATCAGAATTTGCAGACAAAACTTCTTCCATTCAAAGATTAGACACTACTTCTCCCCTTACAACTGTCAGCAAAATGTTACCGAATTCTGCTGGTAGTAACAGGGAGTCAAAGTCTGAAATAATTGGAGGCAGGAAGACTCCTAAGTTAAAAGGTTCTGTTAAAAAGGGAAAGGTTCATGCTAATCCTCCAAATGGCCTTAAAACTGAGGTGACAGCCAATCGATTACAGGTGCCATCAATAAAACACAAAAAAGTAGAAGGTTCTTCCAATGGACGGTTTGAAGCAGTTCAGGAGAAACTTAATGAGTTGCTGGATGGAGATGGTGGAATAAGCAAAAGAAAAGATGCCACCAAAGGGTACTTGAAGCTTTTGTTTCTCACTGTGGCATCAGGAGATAGAATTAATGGTGAAGCAATTCAAAGCAATCGAGATCTTTCCATGATCCTTGATGCTCTTCTGAAAACAAAATCTAGAGCAGTGCTGAATGATATAATTAATAAAAATGGTTTGCAGATGTTACATAACATTATGAAGCAGTACAGGCATGACTTCAAAAAAATTCCAATACTACGAAAGCTTCTTAAGGTCTTGGAGTTCCTAGAAGCAGGCAAGATTTTGACATATGAACATATTAATGGTGGTCCTCCTTGTCGTGGAATGGAGAGCTTTAGAGAGTCAATGCTTTCTCTGACAGAGCATGAAGACAAACAGGTCCATCAAATTGCTCGAAACTTCCGAGACAGATGGTTTCCCAGACATGCCAGAAAACATGGCTATATGGACAGGGATGATAACAGAGTGGAATCTCACAGAAGTTTCAAGTGTAACAGATTTTCAGCATCACAGAGTTATAGGCATGAACAGGATTTAAAGACTACAGAAGCAAGTGATTGTAGTCAGCAGTCGATGCTTGTGACAACTCCAGTAGATGCTGAAGCCCGGGAAGGCTTTCCTGTGCAGTCTCTAGATGGGGTTGAAACCAAAACAGCTGAAAAGCGTAAGCGCAAAAGCCGATGGGATCAACCAGCCGAAACAAACTCGCATTCTGATGTCGTTATGAGCTCTATTGGTGAAAGCCAGAACATTCATGAGGATGTTCCACCAGGGTTTTCGTGTCCAGTAGGTTCATTAAATGCTTCCCTAAACTCTGGCAATCTTGCCTTGCAAAATGCAAGCCGTTCTGGATGTCCCTCTGATATAATTATTGGTCATCCAAAAGAGAAATTTAACTCTTGCTTGGCTGTCTCATTTGGAATGCCATGGTCTGTTGCCCAGCAATATGGAACTCCTCATGCTGAATTTCCAGAGTGTTGGGTCACTGCACCTGGCATGCCTTTCAACCCATTTCCTCCACTACCCCCGTATCCACGGGACAACAAAGATTGTCAACCTTCTAATACTAATGCTATGATAATTGATCAGCCTGCTGAAGTTGAGCAGGGGGATACCAGCGGTATGGTTAATTGTCGCTCTGATGATATGATTCCCAGCACAACTGGTGTTAACCCTGAAGACTCTAACCTTCTGTTTGAGGACAATAAACACATAAGTAAACGATTGAAGGGTGACTCAAATGATTTGGGAACGAGGTACTTTAGACAGCAGAAAATACACCGACCATGGTTCAAGAGGAATGCATGGAAATGTGATGAGAACAACTCTAGTGGTGATATGTGCAGTATAGATGTAGGAGATGTACCAAAAGAGTCAAAAGTTACTTGTGACGCGGAGGATGCAATCTGTAGAGAGGAGTAA
(2) Amino acid sequence of GmASHH2 a: (2040 aa) SEQ ID NO:5 is shown in
MVEMGSCGRSAAIDNPSEKFIIEQQQHLCLEVQEQEVVSVVQESCLEEEACNVVDSNVELSTVIDGCLGEDRVCSKRCVDVTEGSREGLGLGLVSECENADLLPLEKSAEDDCQNYLGVSCGSIEVPCVNSGSEGRFLDEGNFDLPSGSLTADDSQMYCAQLDEQKDDESWSLITDDSQRHCAQQDEQKDDKSDVLPAAGDDLAVVEGKNDVTGVLADAFSHVHDFRDCEVSLESESMADLLVDCNWQSEQEEIMRNTDPLLNVVEKCDALIVEETDACRQISPTPAMEVPSGAPSGALCTDTEVESTSDQPCDQKDGEETDVCRQISPTLAMEVPSGAFWTDTEVESTSDQPCDPKDGEDQNSTCEEIININSCVKISSSPGCNETVGSSPVVGFPCEPALLDPEYEMKNGMLQIEDDACKLKDCSSEETTNSTFRKPFSPESGLPSVALITNCSAKDVPDQQSKGDDVSIDNNNAVNNQGQMDNDGTEAVEVDCITEGIPLPSQRNSRRTKFGRKTQTKKASRKCKNKTKVTHPNGGMKLNLEAARKKRSCFSKPARSSIWGLIGNIEQFFEQDNELGDGEAVCQELGKARSKPQSGKAVKNGASTTSLGSVQKHSVSTTRVRLKIKFGKEVDLSCSNVLIPESVDGLASASYLGSGSGSQKVAGNADDKISEVVALGHSESFNNDLDKDGFVLNEQVANNPLETTEITEKSYGDAEEPCLAVPPEKVVEALIEPINNKGMDPGTSPDSEVINSIPEVQAGEKHQEDAHHAVLGSSKELNSKLDVTISKRGKNKEKVICSSNCITEDGSQGPHKNSRAKHSKNHRRKKNCRDVVSSLELPTDISKSLSSKELSPESLPLSVETELGGSTEALKVKNHTDVKTSDKPSVDHGFSDSLVAENMLSSARPLERKLPKSLRASKVSKTKSKASDSTGRKKTTAGIRKEKQIKAINKSKVKGKGVSLKVTCEVEDCLHPEENAGNHKLDAVGKIIADDNRVSVNLSNLDMLSGVGYGEQLLSPRNAWVRCDDCHKWRRIPAVLADRIDETNCTWTCKDSSDKAFADCAIPQEKSNAEINAELGLSDASGEEDAYEGSKNFKELEYRPPLVSQESTFTHILTNEFLHRSHKTQTIDEIMVCHCKPSQEGKLGCGDECLNRILNIECVQGTCPCGDRCSNQQFQKHKYASLKWFKCGKKGYGLKAIENVAQGQFLIEYVGEVLDMQAYEARQREYALKGHRHFYFMTLNGSEVIDASAKGNLGRFINHSCDPNCRTEKWMVNGEICIGLFALRDIKKDEELTFDYNYVRVFGAAAKKCYCGSPNCRGYIGGGDPLNAELIVQSDSEEEFPEPVMLTKDGEIEDSVPTPEYFNNVDTQSAKHMLKDRDILDNSTTAIDSDGSLEKERSMNPASAVSLLHSSAEMEDSKGKLQSSVQVEEISQQMEDVTSKPMPAVHQGYEKESEFADKTSSIQRLDTTSPLTTVSKMLPNSAGSNRESKSEIIGGRKTPKLKGSVKKGKVHANPPNGLKTEVTANRLQVPSIKHKKVEGSSNGRFEAVQEKLNELLDGDGGISKRKDATKGYLKLLFLTVASGDRINGEAIQSNRDLSMILDALLKTKSRAVLNDIINKNGLQMLHNIMKQYRHDFKKIPILRKLLKVLEFLEAGKILTYEHINGGPPCRGMESFRESMLSLTEHEDKQVHQIARNFRDRWFPRHARKHGYMDRDDNRVESHRSFKCNRFSASQSYRHEQDLKTTEASDCSQQSMLVTTPVDAEAREGFPVQSLDGVETKTAEKRKRKSRWDQPAETNSHSDVVMSSIGESQNIHEDVPPGFSCPVGSLNASLNSGNLALQNASRSGCPSDIIIGHPKEKFNSCLAVSFGMPWSVAQQYGTPHAEFPECWVTAPGMPFNPFPPLPPYPRDNKDCQPSNTNAMIIDQPAEVEQGDTSGMVNCRSDDMIPSTTGVNPEDSNLLFEDNKHISKRLKGDSNDLGTRYFRQQKIHRPWFKRNAWKCDENNSSGDMCSIDVGDVPKESKVTCDAEDAICREE.
(3) Nucleotide sequence of GmASHH2 b: (6189 bp) SEQ ID NO:6 is shown in
ATGATTGAAATGGGGTCGTGTGGGAGATCTGCTGCCATTGATGATCCCTCAGAAAAGTTTGTAATTGAGCAGCAGCATTTGTGCTCAGAGGTTCAAGAGCAGGTTGTTTCTGTGCAGGAGTCTTGTTTAGAAGAAGAGGCTTACAATGTGGTGGACTCTAATGTTGAGCTCTCCACTGTGACTGATGGTTGTCTGCGAGGGGACCGTGTCAGTTCTGAAGGACGTGTTGATGTTACCGAGGGGTCCGGAGAGGGTTTGGGTTTGGCGAGTGAATGCAAAAATGCTGATTTGTTGCCACTGGAGAAATCTACTCAGGATGATTGTCAGAATTGTTTGGGTGTTTCTTGTGGGAGCATTGAAGTTCTCTGTGTAAACAGTGGCTCAGAGGGAAATTTTCAGGATGAGGGTATCTTTGATCAGCTGTCGGGGTCCTTAACTGCAGATGATTCACAGAGGCGTTGTGCTCAACAGGATGAGCAGAAGGATAATAAGAGTGACGTATTGCCTGCAGCAGGAGATGATTCGGATGTCGTGGAAGGCAAAAATGATGAGACTGGTGTTCTTGCTGATGCTTTCAGTCATGCACTTGATTTCAGGGATTGCGAAGTGTCTTTGGAATCAGAATCCATGGCTGACTTGCTAGTTGATTGCAACCAGCAAAGTGAGCAGGAGAAAATTATGAGAAATCCAGATCCCTTGTTGAATGTAGTTGAAAAATGTGACGCTCTAATTGGGGAGGAAACTGATGCTTGCAGGAAGATATCTCCTACTCCGGCTATGGAAGTGCCATCAGGTGCATTATGTACAGATACAGAAGTAGAGAGCATAAATGATCACCCATGTGATCAGAAAGATGGTGAGGAAACTGATGCTTGCAGGCAGATATCTCCTACTCCGACTATGGAAGTGCCATCAGATGCATTATTTACAGATACAGAAGTAGAGAGCATAAGTGATCAGCCATGTGATCAGAAAGATGGTGAGGAAACTGATGCTTGCAGGAAGATATCTCCTACTCTGGCTATGGAAGTGCCATCAGGGCCACCATCAGGTGCATTATGTACAGATGCAAAAGTAGAGAGCACAAATGATCAGCCATGTGATCAGAAAGATATTGAGGATCAGAATAGTACTTGTGAAGAAAAGTTTAAAGCTTTTGTTGATGAAGAAGTCATCAACATTAATTCATGTATAAAAATATCATCCTCTCTAGATTGCCAGGAGACTGTTGCGAGCTCACCTGTAGTTGGTTTTCCTTGTGAGCCTGCCCTGTTGGATCCAGGGTGTGAAATGAAGAATGACATGCTGCAAATAGAAGATGATTTCTGTAAATTGAAGGACTGTTCCTCAGAGGAAACTACCAACTCTACCTTTAGAAAACCATTTTCTCCTGAATCAGGTCTGCCCTCTGTTGCTTTAATTACCAACTGCTCTGCAAAGGATGTTCTTGATCTCCATTCTAAGGGTGATGATGTCTCCATTAATAATAATAATGCTGTTAACAATCCAGGGCAGATGGATAATGATGGAACAAAAGCTGTTGAAGTTGATTGTATCACTGAAAGCATACCGTTACCTTCTCTAAGGGATAGTCGAAGAACTAAATTTGGCCGTAAGACACAAACCAAAAAGGCTTCAAGAAATTGCAAGAACAAAACCAAGGTGACGCATTCAAATGGGGGTATGAAGCTAAACTTAGAGGCTGCTAGAAAGAAAAGAAGTTGTTTCTCCAAACCAGCTCGTTCTTCTGTCTGGGGATTGATTGGGAATATTGAACAGTTTTTTGAGCAAGATAATGAGCTTGGAGTTGGTGAAGCTGTGTGCCAAGAATTGGGGAAGGCTAGAAGCAAGCGTCAAAGTGGGAAAGCGGTTAAGAATGGTGCAAGTACTACCTCATTGAGTTCAGTACAGAAGTCTTCTGTTTCAACTACTCGTGTTCGTCTGAAGATTAAATTTGGGAAAGAAGTTGATTTAAGTTGCTCAAATGTCTTGATTCCAGAGTCAGTTGATGGTTTGGCTTCTGCTTCTTACTTGGTGTCTGATTCAGGTTCTCAGAAAGTAGCCGGCAATGCTGATGATAAAATTTCTGATGCGGTGGCTTTGGGAAATTCAGAATCTTTCAGTAATGACCTGGGCAAGGATGGTCTTGTTCTAAATGAACAAGTTGCAAATAACCCCTTAGAAACTACTGAAATAACAGAGAAGTCATATGGGGATGCAGAGGAACCTTGTCTTGCTGTTCCTCCTGAGAAGGTGGTTGAAGCATTGATTGAACCTATGAGTAATAAGGGTATGGATCCTGGAACCTCACCTGATTCTGAAGTTATCAATTCAATTCCCGAAGTCCAGATTGGAGAAAGACATCAAGAAGACGTACATCATGCTGTTTTAGGTTCTTCTAAAGAATTAAATTCTAAATTGGATGTTACCATCAGTAAGAGAGGGAAGAAGAAAGAGAAACTTATTTGTTCTGGTAATTGTATCACTGAAGATGGATCACAAGGTCCACGTGGGAATAGTAGAGCTAAGCATTCAAAGAATCACAGACGTAAGAAAAATTGCAGGGATGCATTTAGCTCTTTGGAGTTGCCCACTGAGATAAGCAAATCTGTGACCAGTAAAGAATTGTCCCCAGAATTATTACCCCATTCTGGAGAGACTGAACTTGGAGGCTCCGTCGAGGCTTTGAAAGTTAAAAATCATATGGACGCTAAGACAAGTAATAAACCATCTGTTGACCATGGATTTTCAGATTCCCTGGTTTCTGAGAAAATGCTGTCATCTGCAAGACCTTTGGGGCGTAAACTACCTAAAAGTCTTAGACCTAGTAAAGTTAGCAAGACCAAATCTAAAGCTTCTGACTCATCTGGCAGGAAAAAGACTACTGCTGGTACATGCAAGGAGAAACAGAAAAATCCAATTAATAAGAGCAAAGTCAAGGGAAAAGGTGCCTCTCTTAAAGTTACATGTGAAGTGGAAGATTGCCCACATCCAGAAGCAAATGCTGGAAATCATAAGCTGGATGCTATTGGAAAAATTATTGCTGATGACAACAGAGTATCGGTCAATGTATCTAATTTGGACATGCTGTCTGGTGTTGGTTTTGGGGAGCAACTTCTATCACCCCGTAATGCATGGGTGCGTTGTGATGATTGTCACAAGTGGCGGCGAATTCCAGCTGTGCTTGCAGACCGAATTGATGAAACTAACTGCACCTGGACATGTAAGGACAGCAGTGATAAAGCCTTTGCTGATTGTGCTATCCCTCAAGAAAAGTCTAATGCAGAGATTAATGCGGAGTTGGGATTATCAGATGCCTCGGGTGAAGAAGATGCATATGAAGGTTCCAAAAATTTTAAGGAATTAGAATATTGGCCACCTATAGTTTCCCAGGAGTCAACTTTTACCAATATTTTGACCAATGAATTTCTGCATCGTAGCCATAAAACTCAGACTATTGACGAGATAATGGTATGTCATTGCAAGCCATCTCAAGGAGGAAAGTTAGGCTGTGGGGATGAATGTCTAAATCGGATTCTTAATATCGAATGTGTACAAGGAACCTGCCCATGTGGGGACCGTTGTTCCAACCAGCAGTTCCAAAAACACAAGTATGCTAGCCTGAAGTGGTTTAAATGTGGGAAAAAGGGTTACGGACTGAAGGCAATTGAGGATGTAGCTCAAGGCCAGTTTCTTATTGAATACGTTGGAGAGGTGCTTGATATGCAAACGTATGAGGCACGGCAAAGAGAGTACGCTTTGAAGGGCCATCGGCATTTCTATTTTATGACCTTGAATGGTAGTGAGGTGATAGATGCAAGTGCAAAAGGAAACTTGGGGCGTTTCATTAATCATAGTTGTGATCCTAATTGTCGGACAGAAAAGTGGATGGTGAATGGGGAAATCTGTATTGGACTGTTTGCATTGAGGAATGTTAAGAAGGATGAAGAATTGACATTCGATTACAACTATGTAAGGGTTTTTGGTGCTGCTGCCAAAAAATGCTATTGTGGTTCGTCTAACTGTCGAGGTTATATAGGTGGTGGTGATCCACTTAATGCTGAATTGATAGTTCAAAGTGATTCAGAAGAAGAATTTCCCGAACCTGTTATGCTTACCAAAGATGGTGAAATTGAAGATGCTGTACCTACACCCAAGTATTTTAATAATGTTGATACAGAATCTGCTAAACATATGTTGAAAGACAGGGATATATTGGACAATCCTACAACTGCTATAGATTCAGATGGTTCTCCAGAGAAAGAGAGTTCTATGAACCCTGCCTCTGCCATTTCTCTGTTGCACAGCTCAGCAGAAATGGAGGATTCAAAGGGTAAATTACCATCTTCTGTTAGAGATGAAGAAATTTCTCAACAAATGGAGGATGTAACAAGCAAACCCATGCCTTCTGTACACCAAGGATATGAAAAGGAATCAGAGTTTGCAGACAAAACTTCTTCCATTCAAAGATTAGAGACTACTTCTCCCCCTACAACTGTCAGCAAAATGTTACCGAATTCTGCTGGTAGTAATAGGGAGTCCAAGTCTGAAATAATTGGAGGCAAGAAGACTCCTAAGTTAAATGGTTCTGTTAAAAAGGGGAAGGTTCATGCTAATCCTCCAAATGGCCTTAAAACTGAGGTGACAGCCAATCGATTACAGGTGTCATCTATAAAACACAAAAAAGTAGAAGGTTCTTCCAATGGACGGTTTGAAGCAGTTCAGGAGAAACTTAATGAGTTGCTGGATGGAGATGGTGGAATAAGCAAAAGAAAAGATGCCACCAAAGGGTACTTGAAGCTTTTGTTTCTCACTGTGGCATCAGGTGATAGAATTAATGGTGAAGCTATTCAAAGCAATCGAGATCTTTCCATGATCCTTGATGCCCTTCTGAAAACAAAATCTAGAGCAGTGCTGAATGATATAATTAACAAAAATGGTTTACAGATGTTACATAACATTATGAAGCAGTACAGGCATGACTTCAAAAAAATTCCAATACTACGAAAGCTTCTTAAGGTCTTGGAGTTCCTAGAAGCAAGCAAGATTTTGACATCTGAACATATTAATGGTGGTCCTCCTTGTCATGGAATGGAGAGCTTTAGAGAGTCAATGCTTTCTCTGACAGAGCATGAAGACAAACAGGTCCATCAAATTGCTCGAAACTTCCGAGACAGATGGTTTCCCAGACACGCCAGAAAACATGGCTATATGGACAGGGATGATAACAGAGTGGAATCTCACAGAAGTTTCAAGTGCAACAGATTTTCAGCATCACACAGTCAGAGGCATGAACAGGATTTAAGGACTACAGAAGCAATTGATTGTAGTCAGCAGGCGATGCTTGTGACAACTCCAGTAGATGCTGAAACCTGGGAAGGCTGTCCTGTGCAGTCTCTAGATGGGGTTGAAATCAAAAGAGCTAAAAAACGCAAGCGCAAAAGCCGATGGGATCAACCAGCCGATACAAACTCGCATTCTGATGCCGTTATGAGCTCTATCGGTGAAAGCCAGAACATTCCCGAGGATGGTCCACCAGGGTTTTCATGTCCAGTAGGTTCATTAAATGCTTCCCTAAACTCTGGTAATCTTGCCTTGCAAAATGCAAGCCGTTCTGGATGCCCCTCTGATATAGTTATTGGTCATCCAAAAGAGAAATTTAACTCTCACTTGCCTGTCTCATATGGAATGCCATGGTCTGCCCAGCAATATGGAACACCTCATGCTGAATTTCCTGAGTGTTGGGTCACTGCACCTGGCATGCCTTTCAACCCATTTCCTCCACTACCCCCGTATCCACGGGACAACAAAGACTGTCAACCTTCTAATACTACTAATGCTATGATAATTGATCAGCCTGCTGAAGTTAAGCAGGGGGATACCAGTGGTATGGTTAATTGTTGCTCAGATGATATGATTCCCAGCACAACTGGTGTTAACTCTGAAGACTCCAACCTACTGTTTGAGGACGATAAACACATAAGTAAACGATTGAAGGGTGATTCCAATGATTTGGGAACGAGGTACTTTAGACAGCAGAAAATACACCGACCATGGTTCAAGAGGAATGCATGGAAATGTGATGAGAACAACTCTTGTGGTGATATGTGCAGTATAGATGTAGGAGATGTACCAAAAGAGTCAAAAGTTACTTGTGACGCGGAGGATGCAATCTGTAGAGAGGAGTAA
(4) Amino acid sequence of GmASHH2 b: (2062 aa) SEQ ID NO:7 is shown in
MIEMGSCGRSAAIDDPSEKFVIEQQHLCSEVQEQVVSVQESCLEEEAYNVVDSNVELSTVTDGCLRGDRVSSEGRVDVTEGSGEGLGLASECKNADLLPLEKSTQDDCQNCLGVSCGSIEVLCVNSGSEGNFQDEGIFDQLSGSLTADDSQRRCAQQDEQKDNKSDVLPAAGDDSDVVEGKNDETGVLADAFSHALDFRDCEVSLESESMADLLVDCNQQSEQEKIMRNPDPLLNVVEKCDALIGEETDACRKISPTPAMEVPSGALCTDTEVESINDHPCDQKDGEETDACRQISPTPTMEVPSDALFTDTEVESISDQPCDQKDGEETDACRKISPTLAMEVPSGPPSGALCTDAKVESTNDQPCDQKDIEDQNSTCEEKFKAFVDEEVININSCIKISSSLDCQETVASSPVVGFPCEPALLDPGCEMKNDMLQIEDDFCKLKDCSSEETTNSTFRKPFSPESGLPSVALITNCSAKDVLDLHSKGDDVSINNNNAVNNPGQMDNDGTKAVEVDCITESIPLPSLRDSRRTKFGRKTQTKKASRNCKNKTKVTHSNGGMKLNLEAARKKRSCFSKPARSSVWGLIGNIEQFFEQDNELGVGEAVCQELGKARSKRQSGKAVKNGASTTSLSSVQKSSVSTTRVRLKIKFGKEVDLSCSNVLIPESVDGLASASYLVSDSGSQKVAGNADDKISDAVALGNSESFSNDLGKDGLVLNEQVANNPLETTEITEKSYGDAEEPCLAVPPEKVVEALIEPMSNKGMDPGTSPDSEVINSIPEVQIGERHQEDVHHAVLGSSKELNSKLDVTISKRGKKKEKLICSGNCITEDGSQGPRGNSRAKHSKNHRRKKNCRDAFSSLELPTEISKSVTSKELSPELLPHSGETELGGSVEALKVKNHMDAKTSNKPSVDHGFSDSLVSEKMLSSARPLGRKLPKSLRPSKVSKTKSKASDSSGRKKTTAGTCKEKQKNPINKSKVKGKGASLKVTCEVEDCPHPEANAGNHKLDAIGKIIADDNRVSVNVSNLDMLSGVGFGEQLLSPRNAWVRCDDCHKWRRIPAVLADRIDETNCTWTCKDSSDKAFADCAIPQEKSNAEINAELGLSDASGEEDAYEGSKNFKELEYWPPIVSQESTFTNILTNEFLHRSHKTQTIDEIMVCHCKPSQGGKLGCGDECLNRILNIECVQGTCPCGDRCSNQQFQKHKYASLKWFKCGKKGYGLKAIEDVAQGQFLIEYVGEVLDMQTYEARQREYALKGHRHFYFMTLNGSEVIDASAKGNLGRFINHSCDPNCRTEKWMVNGEICIGLFALRNVKKDEELTFDYNYVRVFGAAAKKCYCGSSNCRGYIGGGDPLNAELIVQSDSEEEFPEPVMLTKDGEIEDAVPTPKYFNNVDTESAKHMLKDRDILDNPTTAIDSDGSPEKESSMNPASAISLLHSSAEMEDSKGKLPSSVRDEEISQQMEDVTSKPMPSVHQGYEKESEFADKTSSIQRLETTSPPTTVSKMLPNSAGSNRESKSEIIGGKKTPKLNGSVKKGKVHANPPNGLKTEVTANRLQVSSIKHKKVEGSSNGRFEAVQEKLNELLDGDGGISKRKDATKGYLKLLFLTVASGDRINGEAIQSNRDLSMILDALLKTKSRAVLNDIINKNGLQMLHNIMKQYRHDFKKIPILRKLLKVLEFLEASKILTSEHINGGPPCHGMESFRESMLSLTEHEDKQVHQIARNFRDRWFPRHARKHGYMDRDDNRVESHRSFKCNRFSASHSQRHEQDLRTTEAIDCSQQAMLVTTPVDAETWEGCPVQSLDGVEIKRAKKRKRKSRWDQPADTNSHSDAVMSSIGESQNIPEDGPPGFSCPVGSLNASLNSGNLALQNASRSGCPSDIVIGHPKEKFNSHLPVSYGMPWSAQQYGTPHAEFPECWVTAPGMPFNPFPPLPPYPRDNKDCQPSNTTNAMIIDQPAEVKQGDTSGMVNCCSDDMIPSTTGVNSEDSNLLFEDDKHISKRLKGDSNDLGTRYFRQQKIHRPWFKRNAWKCDENNSCGDMCSIDVGDVPKESKVTCDAEDAICREE.
1.2 Gene knockout target site predictive analysis
GmASHH2a (Glyma.06G117700), gmASHH2b (Glyma.04G24540) gene sequences were downloaded in soybean database (https:// phytozome-next. Jgi. Doe. Gov /). A website (http:// CRISPR. Hzau. Edu. Cn/CRISPR2 /) is utilized to carry out predictive analysis on a potential target of 19nt + PAM (NGG) on the GmASHH2a/b gene.
The nucleotide sequence of the sgRNA1 is shown in SEQ ID NO:8 (CTTCGGCAGATTTCTCCAGTGG);
the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO:9 (CCTGAGTATTCTCCAGTGG).
1.3GmASHH2a/b gene knockout vector, genetic transformation and screening and identification of transgenic material
Construction of 1.3.1GmASHH2a/b gene knockout vector
The method for constructing the recombinant vector containing the sgRNA composition includes a method for constructing pCBSG015-GmASHH2s, and preferably includes the following steps:
A. <xnotran> + GmASHH2a/b 2 sgRNA ( : aagctt (HindIII) cgttgaacaacggaaactcgacttgccttccgcacaatacatcatttcttcttagctttttttcttcttcttcgttcatacagttttttt ttgtttatcagcttacattttcttgaaccgtagctttcgttttcttctttttaactttccattcggagtttttgtatcttgtttcatagtttgtcccaggattagaatgattaggcatcgaaccttcaagaatttgattgaataaaacatcttcattcttaagatatgaagataatcttcaaaaggcccctgggaatctgaaagaagagaagcaggcccatttatatgggaaagaacaatagtatttcttatataggcccatttaagttgaaaacaatcttcaaaagtcccacatcgcttagataagaaaacgaagctgagtttatatacagctagagtcgaagtagtgatt (AtU 6 promoter) gCTTCGGCAGATTTCTCCAG (Target) gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SgRNA) tttttt gttttacgttgaacaacggaaactcgacttgccttccgcacaatacatcatttcttcttagctttttttcttcttcttcgttcatacagtttttttttgtttatcagcttacattttcttgaaccgtagctttcgttttcttctttttaactttccattcggagtttttgtatcttgtttcatagtttgtcccaggattagaatgattaggcatcgaaccttcaagaatttgattgaataaaacatcttcattcttaagatatgaagataatcttcaaaaggcccctgggaatctgaaagaagagaagcaggcccatttatatgggaaagaacaatagtatttcttatataggcccatttaagttgaaaacaatcttcaaaagtcccacatcgcttagataagaaaacgaagctgagtttatatacagctagagtcgaagtagtgatt (AtU 6 promoter) gCCTGAGTAGATTTCTCCAG (Target) gttt tagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc (SgRNA) tttttgttttagagctt cggctagtccgtagcgcgtgcgccaattctgcagacaaatggccccggg (SmaI) (SEQ ID NO:10 ). </xnotran>
B. And (3) linking the 2 expression cassettes with a pCBSG015 linear plasmid obtained by enzyme digestion of HindIII and SmaI to obtain a pCBSG015-GmASHH2s recombinant vector.
The vector used in the present invention is pCBSG015 (Basta) provided by Mimi Biotech Ltd, the resistance in prokaryotes is Kanamycin (Kanamycin), and the resistance in eukaryotes is glufosinate-ammonium (Basta; PPT). Vector construction is schematically shown in FIG. 1.
The pCBSG015-GmASHH2s recombinant vector was introduced into EHA105 Agrobacterium. Adding 2 mu L of pCBSG015-GmASHH2s recombinant vector into the initially fused EHA105 agrobacterium infected cell, performing ice bath for 5min, performing liquid nitrogen quick freezing for 1min, performing water bath heat shock at 37 ℃ for 3min, and performing ice bath for 3min. 1mL of LB liquid medium was added thereto, and shaking culture was performed at 28 ℃ for 90min. Draw 200. Mu.L of the bacterial suspension and smear on LB + Kan + Rif + Gen plate. Culturing in 28 deg.C incubator for 48h. Selecting a single colony, and performing colony PCR (polymerase chain reaction) as a PCR system: 1 μ L of template DNA; 2 × Rapid Taq Master Mix 7.5 μ L; forward and reverse primers are 0.5 mu L respectively; ddH 2 O5.5. Mu.L. The reaction conditions for PCR amplification were as follows: performing pre-denaturation at 98 ℃ for 5min; 30sec at 98 ℃, 30sec at 60 ℃, 3sec at 72 ℃,30 cycles; extension at 72 ℃ for 5min.
1.4 genetic transformation of Soybean
(1) Seed disinfection
The seeds were placed in a sterilized 100mL triangular flask, and 75% alcohol was added thereto, followed by rinsing 2 times. Adding 25mL 84 disinfectant, 75mL sterilized water and 3 drops of Tween-20, and soaking for 15min. After three times of sterilization water washing, the seeds are placed on a germination culture medium (GM) with the umbilicus facing downwards for germination for 1-2 d under illumination (26 ℃,18h illumination/6 h dark light cycle).
Germination Medium (GM): 1/2MS salt ion +20g/L sucrose +7g/L agar, pH 5.8, autoclaving.
(2) Preparation of Agrobacterium liquid
Positive monoclonals were picked and shake-cultured (250rpm, 28 ℃) in 2mL YEP medium containing antibiotics to saturation of growth (about 12 h). Then 0.2mL of saturated bacterial liquid is added into 250mLYEP culture medium containing antibiotics for overnight culture till logarithmic growth phase (OD) 650 =0.3 to 0.6). Colonies were collected by centrifugation and the pellet resuspended to OD using liquid Coculture Medium (CM) 650 Is 0.6.
YEP medium: 5g/L NaCl, 5g/L yeast extract, 10g/L peptone and 15g/L agar, and autoclaving.
(3) Preparation of explants
30ml of Agrobacterium solution was poured into a petri dish. A longitudinal cut was made along the hilum with a scalpel blade to separate the cotyledons and remove the seed coat. The embryonic axis at the junction of the hypocotyl and cotyledon is excised to obtain a half-seed explant. 50 explants were prepared per Agrobacterium plate. Ensuring that the whole explant is suspended by the bacterial liquid. After 30min of padding, explants were transferred to solid coculture medium with sterilized forceps, 15 explants per dish, placed horizontally. The Petri dishes were sealed with a breathable tape and transferred to an incubator (23 ℃ C.), and dark culture was carried out for 3-5 days.
co-Cultivation Medium (CM): 1/2MS salt ion +3.9g/L MES +30g/L sucrose + B5 vitamin +153mg/L DTT +2mg/L zeatin +40mg/L AS +7g/L agar, pH 5.4, autoclaved.
(4) Stem induction and selection
Explants were placed on SI medium without selection (15 explants per dish). The petri dish was sealed with a gas permeable tape and incubated with light for 7d (26 ℃,18h light/6 h dark photoperiod). The elongated cotyledon hypocotyls were then excised and the explant tissue was replaced with SI medium containing the selection agent and grown in light culture for 21d (7 explants per dish).
Stem induction medium (SI): b5 salt ion +0.98g/L MES +30g/L sucrose + B5 vitamin +150mg/L cefuroxime +450mg/L temustine +1 mg/L6-BA +50mg/L asparagine +50mg/L glutamine +6mg/L glufosinate +7g/L agar, pH 5.7, and autoclaving.
(5) Elongation of the stem
The differentiated explants were transferred to SE medium. Cotyledons were excised from the explants and a new incision was made at the base of the developing node. Explants were transferred to fresh Stem Elongation (SE) medium and incubated with light at 26 ℃ for 2-8 week. Fresh SE medium was changed every 2 weeks. A fresh horizontal incision was made at the base of the outer implant for each media change.
Stem Elongation (SE) medium: MS salt ion +0.6g/L MES +30g/L sucrose + B5 vitamin +150mg/L cefuroxime +450mg/L temustine +0.1mg/L IAA +0.5mg/L GA +1mg/L zeatin +50mg/L asparagine +50mg/L glutamine +6mg/L glufosinate +7g/L agar, pH 5.7, and autoclaving.
(6) Rooting
When the stems grew to 3cm long, they were cut from the tissue, immersed in IBA (1 mg/ml) for 1-2 min, and then transferred to a glass flask containing Rooting Medium (RM) for further culture. After 2 weeks, when more than 2 shoots grew, they were transplanted into soil and acclimatized. Culturing at 26 deg.C for about 1 week to make the seedling survive.
Rooting Medium (RM): 1/2MS salt ion, 0.6g/L MES, 20g/L sucrose, B5 vitamin, 3mg/L glufosinate-phosphine, 7g/L agar, pH 5.7, and autoclaving.
1.5 screening and characterization of transgenic Material
Extracting DNA of resistant plants obtained by tissue culture, and carrying out PCR amplification on the DNA by using corresponding upstream and downstream primers of a carrier, wherein the plants capable of amplifying target segment bands are transgenic plants. In the invention, the positive detection method preferably adopts a forward primer Cas9-detection F (CCGACGAGTACAAGGTGCCACCA) and a reverse primer Cas9-detection R (GATCAGATTTTCCAGCCATCTCTC) to carry out PCR amplification detection (the electrophoresis detection result is shown in figure 2), the transgenic plant with the target amplification product is inserted with the T-DNA fragment, and the transgenic plant without the target product is not inserted with the T-DNA fragment. The reaction conditions for the PCR amplification are preferably as follows: pre-denaturation at 98 ℃ for 5min; 30sec at 98 ℃, 30sec at 60 ℃, 3sec at 72 ℃,30 cycles; extension at 72 ℃ for 5min.
1.6 Gene editing type detection of transgenic Material
Extracting DNA of a positive plant, and detecting the quality of the DNA by using a primer GmActin F (CGGTGGTTCTATCTTGGCATC)/GmActinR (GTCTTTCGCTTCAATAACCCTA) PCR, wherein the reaction system is as follows: 1 mu L of template DNA; 2 × Rapid Taq Master Mix 7.5 μ L; the upstream primer and the downstream primer are respectively 0.5 mu L; ddH 2 O5.5. Mu.L. The PCR reaction conditions are preferably: pre-denaturation at 98 ℃ for 5min; 30sec at 98 ℃, 30sec at 56 ℃, 30sec at 72 ℃ and 30 cycles; extension at 72 ℃ for 5min. The electrophoresis pattern of the PCR reaction product is shown in FIG. 3.
The primer J206141-F1 (TTGAGCAGCAGCATTTG)/J206141-R1 (TCCTGCTCACTTTGCCAGTT) at 225bp upstream and downstream of the target site of the GmASHH2a gene performs PCR amplification on the DNA of the primer, and the PCR product is sent to sequencing. The reaction system is preferably as follows: form panelDNA 1. Mu.L, 2 × Rapid Taq Master Mix 7.5. Mu.L, upstream and downstream primers 0.5. Mu.L each, ddH 2 O5.5 μ L; the reaction conditions are preferably: pre-denaturation at 98 ℃ for 5min; 30sec at 98 ℃, 30sec at 58 ℃, 30sec at 72 ℃ and 30 cycles; extension at 72 ℃ for 5min. The electrophoresis results of the PCR reaction products are shown in FIG. 4.
The DNA of the GmASHH2b gene is subjected to PCR amplification by a primer J206141-F3 (GCAGGTTGTTTCTGTGCAGG)/J206141-R3 (TCTCCTGCTCACTTTGCTGG) 176bp upstream and downstream of the target site, and the PCR product is sent to sequencing. The reaction system is preferably as follows: template DNA 1. Mu.L, 2 × Rapid Taq Master Mix 7.5. Mu.L, upstream and downstream primers 0.5. Mu.L each, ddH 2 O5.5 μ L; the reaction conditions are preferably: performing pre-denaturation at 98 ℃ for 5min; 30sec at 98 ℃, 30sec at 58 ℃, 30sec at 72 ℃ and 30 cycles; extension at 72 ℃ for 5min. The results of electrophoresis of the PCR reaction products are shown in FIG. 5.
2 results of
2.1 construction of CRISPR/Cas9 Gene knockout vector and Soybean genetic transformation
With cloned GmASHH2a and GmASHH2b as target genes, a segment of 19nt + PAM (NGG) which is positioned in the 1 st exon of the GmASHH2a and GmASHH2b genes and has the GC content of 55% is selected as a target sequence, and a CRISPR/Cas9 gene editing target site is shown in a figure 6. By referring to a CRISPR/Cas9 vector construction process and system, a CRISPR/Cas9 gene knockout vector pCBSG015-35S (GmASHH 2a/GmASHH2b-Cas 9) is successfully constructed, and is respectively transformed into Agrobacterium tumefaciens EHA105, and a positive monoclonal is selected for genetic transformation of Tianlongyi (the soybean genetic transformation process is shown in figure 7).
2.2 acquisition and identification of CRISPR/Cas9 Gene editing Material
Through sequencing comparison analysis (specific results are shown in fig. 8), 6 transgenic plants in which genes of GmASHH2a or GmASHH2B are edited are obtained from 10T 1-generation CRISPR/Cas9 knockout plants, wherein the genes of the GmASHH2a in the plants with the numbers of A012a, A034a, A035a, A037a and B016a are edited; and the genes GmASHH2B were edited in the plants numbered A012a, A034a, A035a, A037a and B026 a. The soybean CRISPR/Cas9 gene editing material with numbers B016a and B026a is probably a mutant plant with single gene editing of GmASHH2a or GmASHH 2B; and the materials numbered a012a, a034a, a035a, and a037a are mutant plants with double gene editing of GmASHH2a and GmASHH2 b.
2.3 acquisition and identification of homozygous mutant Material for CRISPR/Cas9 Gene editing
Carrying out backcross, selfing and sequencing comparison analysis work on progeny single plants of the identified CRISPR/Cas9 gene editing plants, and finding that the number of the plants is A037a-4, wherein the GmASHH2a and GmASHH2b genes are effectively edited and are homozygous mutation, wherein the GmASHH2a gene lacks 218bp (-218 bp deletion), so that the reading frame of the gene is shifted and mutated, and translation is terminated when meeting a termination codon TGA at the 28 th base behind a PAM locus; and the GmASHH2b gene is deleted by 10bp (-10 bp deletion), and translation is terminated early when meeting a termination codon TAA at the 64 th base behind the PAM site. Meanwhile, the GmASHH2a and GmASHH2b genes in the A012a-3 plant are also edited effectively and are homozygous mutations, wherein 218bp of the GmASHH2a gene is deleted (same as A037 a-4); the GmASHH2b gene is deleted for 134bp (-134 bp deletion), and translation is terminated when meeting a termination codon TGA at the 13 th base after the PAM locus (CRISPR/Cas 9 gene editing plant mutation type analysis is shown in figure 9).
2.4 field phenotypic Observation and analysis of CRISPR/Cas9 Gene editing Material
Through laboratory and field phenotype observations of CRISPR/Cas9 gene editing homozygous mutant plants, it was found that the laboratory phenotype results are shown in fig. 10 and the field phenotype is shown in fig. 11, both homozygous mutant materials a012a-3 and a037a-4 exhibit a late-flowering phenotype compared to wild-type WT (aroun one), wherein a012a-3 flowers 13d later than the control under long-day conditions (spring sowing); a037a-4 flowering at 11 d; under short-day conditions (fall sowing), A012a-3 and A037a-4 flowered 5d later compared to the control (as shown in Table 1). Compared with wild WT (Tianlong I), the homozygous mutant materials A012a-3 and A037a-2, -3, -4, -5 show short plants, shortened internodes, increased main stem nodes and the like in vegetative growth stage; the reproductive growth stage is manifested by late blossoming, reduced weight per hundred grains, increased number of effective branches, etc. (statistical analysis results are shown in fig. 12 and table 2).
TABLE 1 transgenic lines field Primary traits
TABLE 2 comparison of yield-constituting factors of transgenic lines
Claims (7)
- The application of GmASHH2a/b protein in regulating and controlling the growth and development of plants is characterized by comprising at least one of the following components:(1) The GmASHH2a/b protein is applied to controlling the flowering phase of plants;(2) The GmASHH2a/b protein is applied to increasing the effective pod number of a plant single plant;(3) The GmASHH2a/b protein is applied to increasing the number of main stem nodes of plants;(4) The GmASHH2a/b protein is applied to increasing effective branches of plants;the amino acid sequence of the GmASHH2a protein is shown as SEQ ID NO. 5; the amino acid sequence of the GmASHH2b protein is shown as SEQ ID NO. 7.
- 2. The use according to claim 1, wherein the GmASHH2a gene encoding the GmASHH2a protein has a nucleotide sequence shown in SEQ ID NO. 4; the nucleotide sequence of the GmASHH2b gene for coding the GmASHH2b protein is shown as SEQ ID NO. 6.
- 3. The use of claim 1, wherein the plant is soybean.
- 4. The use of claim 2, wherein said controlling plant flowering is delayed soybean flowering.
- 5. A method of growing soybeans, comprising the steps of:(1) Constructing a GmASHH2a/b gene knockout vector;(2) Carrying out soybean genetic transformation;(3) Screening and identifying transgenic materials.
- 6. The method according to claim 5, wherein the GmASHH2a/b gene knockout vector comprises sgRNA1 and sgRNA2; the sgRNA1 is shown as SEQ ID NO:8 is shown in the specification; the nucleotide sequence of the sgRNA2 is shown in SEQ ID NO: shown at 9.
- 7. The method of claim 6, wherein the vector is pCBSG015.
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