Application of GhSTM gene in improvement of genetic transformation efficiency of cotton

Abstract

The invention discloses application of a GhSTM gene in improvement of genetic transformation efficiency of cotton. According to the invention, a novel GhSTM gene is cloned from cotton, an overexpression vector is constructed by using a DNA sequence of a CDS full-length fragment of the gene, then agrobacterium tumefaciens is introduced, the cotton is transformed by using an agrobacterium tumefaciens-mediated method, and in the process, it is found that compared with a transformed WMV067 empty vector, the overexpression GhSTM can screen out more green buds, improve the regeneration efficiency and shorten the transformation time. Meanwhile, the agrobacterium of the over-expressed GhSTM gene can greatly improve the transformation efficiency of the cotton, and especially solves the problem of genotype limitation of some main cultivated cotton varieties with low transformation efficiency. Therefore, overexpression of the gene and application of the gene to commercial cotton varieties difficult in genetic transformation contribute to improvement of the genetic transformation efficiency of the commercial cotton varieties, so that the yield, the quality and the stress resistance of high-quality cotton germplasm are improved, and the gene has important economic value and social benefit.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and more specifically, relates to an application of a GhSTM gene in improving cotton genetic transformation efficiency. Background Art

[0002] Cotton is one of the most important economic crops in the world. my country is both the world's largest cotton producer and the largest consumer. The development of cotton production is closely related to the selection and promotion of new varieties. The currently promoted varieties or the direction of variety selection can no longer meet the development needs of the cotton textile industry and cotton production. Genetic engineering technology has opened up a new path for increasing plant yield, improving quality and enhancing resistance. Efficient regeneration and genetic transformation systems are the basis and core of plant genetic engineering. People have tried to improve cotton using different transformation methods, such as gene gun-mediated, Agrobacterium-mediated, and pollen tube channels, and cultivated new varieties and materials of insect-resistant and herbicide-resistant cotton. Transgenic cotton not only increases agricultural product yield, improves quality, and increases farmers' income, but also reduces the use of pesticides and protects the environment and biodiversity. However, cotton is a crop that is difficult to genetically transform. Global transgenic cotton research and industrialization are obviously lagging behind. Inefficient genetic transformation technology is one of the bottlenecks restricting the research and development of transgenic cotton.

[0003] The genetic transformation efficiency of cotton has always been low, but great progress has been made in recent years. The Cotton Research Institute of the Chinese Academy of Agricultural Sciences has created a new "stem cell-based" cotton transgenic method, which breaks the limitations of traditional transgenic methods and can be used to perform transgenic operations on any cotton variety, quickly obtain cotton transgenic engineering materials, and to a certain extent break the genotype dependence of cotton transformation. Despite this, the genotype dependence of cotton is still very serious. The transformation efficiency of some commercial varieties such as Yuanmian No. 8, Xinlu Zao series, Tahe No. 2, and Sea Island cotton and Asian cotton is still low, far from meeting the needs of cotton breeding. Therefore, solving the genotype limitation of genetic transformation of all varieties of cotton, especially improving the transformation efficiency of the main cotton varieties, is urgent to give full play to the role of transgenic technology in cotton genetic breeding.

[0004] Although many tissue culture programs and technologies have achieved certain results in improving cotton genetic transformation, it is clear that they cannot solve the problem of cotton genotype limitation. Plant tissue culture regeneration performance, as a key trait affecting the success or failure of transgenic technology, is bound to be controlled by one or more major effect genes. Therefore, cloning and utilizing regeneration-related genes, screening regeneration key factors, and developing broad-adaptable molecular tools that can improve crop regeneration ability and genotype-independent genetic transformation have become the key to solving the genotype dependence of cotton genetic transformation. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide an application of the GhSTM gene in promoting the genetic transformation efficiency of cotton.

[0006] In order to achieve the above-mentioned object, the technical solution of the present invention is, on the one hand, to provide the application of GhSTM gene in improving the genetic transformation efficiency of cotton.

[0007] Furthermore, the nucleotide sequence of the GhSTM gene is shown in SEQ ID NO.3.

[0008] Furthermore, the GhSTM gene was amplified using the 49 genome of the China Cotton Research Institute as a template through the primers shown in SEQ ID NO.1 and SEQ ID NO.2.

[0009] Furthermore, the amino acid sequence encoded by the GhSTM gene is shown in SEQ ID NO.4.

[0010] Furthermore, the GhSTM gene was constructed onto the cotton vector WMV067 and transformed into cotton, thereby enhancing the expression of the GhSTM gene in the cotton material and thus improving the genetic transformation efficiency of cotton.

[0011] On the other hand, the technical solution of the present invention is to provide a recombinant expression vector and a genetically engineered bacterium containing the GhSTM gene.

[0012] On the other hand, the technical solution of the present invention is to provide a recombinant expression vector containing the GhSTM gene and a genetically engineered bacterium for use in improving the genetic transformation efficiency of cotton.

[0013] On the other hand, the technical solution of the present invention is to provide a protein encoded by the GhSTM gene.

[0014] On the other hand, the technical solution of the present invention is to provide the application of the protein encoded by the GhSTM gene in improving the genetic transformation efficiency of cotton.

[0015] On the other hand, the technical solution of the present invention is to provide the application of GhSTM gene in cotton breeding.

[0016] Beneficial effects of the present invention:

[0017] The present invention clones a novel GhSTM gene from cotton and verifies its function of improving genetic transformation efficiency in cotton. The present invention constructs an overexpression vector with the DNA sequence of the full-length fragment of the CDS of the gene, then introduces Agrobacterium, and transforms cotton by Agrobacterium-mediated method. In the process, it is found that compared with the transformation of the WMV067 empty vector control, overexpression of GhSTM can screen out more green buds, improve regeneration efficiency, improve the efficiency of obtaining positive plants from the stem apical meristem, and shorten the transformation time. Therefore, overexpression of the gene and application to commercial cotton varieties that are difficult to genetically transform can help improve their genetic transformation efficiency, thereby improving the yield, quality and stress resistance of high-quality cotton germplasm, and has important economic value and social benefits.

[0018] The present invention provides the application of GhSTM gene in genetic transformation of upland cotton. The present invention constructs WMV067-GhSTM overexpression vector, transforms Xinjiang main cultivated variety Yuanmian No. 8 with serious genotype restriction by Agrobacterium-mediated method, and the transformation efficiency is increased from original 1.20-1.82% to 7.14-7.92%, the Sea Island cotton Xinhai 78 variety is increased from original 0.50-0.95% to 1.08-2.50%, and the Asian cotton stone system Asia No. 1 is increased from original 0.91-1.30% to 2.27-2.61%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the WMV067-GhSTM overexpression vector structure.

[0020] Figure 2 PCR detection results of STM gene in transgenic cotton plants. In the figure, 1-20 are transgenic plants; CK is the non-transgenic control; P is the plasmid positive control; M is the markerIII molecular weight standard.

[0021] Figure 3 This is the germination status of GhSTM gene-transfected material and empty vector-transfected control material. DETAILED DESCRIPTION

[0022] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments.

[0023] Example 1: GhSTM gene cloning

[0024] According to the sequence of the GhSTM gene in cotton (>Gh_A06G1334.1), primers were designed: GhSTM-F: 5'-GAGAGAACACGGGGGACGTCGACATGGAAGGTGGTTC-3' (SEQ ID NO.1) GhSTM-R: 5'-GAACATCGTATGGGTACATGGATCCGAGAAGCGTTGGAGAG-3' (SEQ ID NO.2)

[0025] Then, RNA of cotton variety Zhongmiansuo 49 (a highly efficient transgenic recipient variety for cotton stem tip transformation) was extracted and reverse transcribed into cDNA as a template. PCR amplification was performed with the above primers to obtain a PCR product of 1128 bp fragment, i.e., the GhSTM gene with restriction sites (the GhSTM gene sequence is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4), which was set aside. Then, the WMV067 vector (sequence is shown in SEQ ID NO.5) was digested with SalI and BamHI restriction endonucleases to obtain a linearized vector. The above PCR product and the above linearized vector were subjected to a ligation reaction, and the ligation product was transformed into competent Escherichia coli. After resistance screening, a single clone was selected for sequencing, and the correctly sequenced vector was named WMV067-GhSTM (structure is shown in Figure 1 As shown). WMV067-GhSTM was transformed into Agrobacterium competent EHA105, and positive clones were selected for use. At the same time, WMV067 (WMV067-GFP) empty vector was transformed into Agrobacterium competent EHA105 using the above method, and positive clones were selected for use.

[0026] The gene sequence of GhSTM is:

[0027] ATGGAAGGTGGTTCCAGTAGCACTTCTTGCATGATGGCTTTTGGACACAATAGTAATGGA

[0028] CTGTGTCCCATGACGATGATGCATCATCCCATGACTTCTCATCTTCATCCTCAACATCAAC

[0029] ATCAACAACATCATCATCATCCTAATTCTGGCTCAAGTCCCTTATTTCTTCCCCAACCTCC

[0030] CACCAACAATCAAGATCAGAACCACAATAGCAGCAGTGGATCCTCTATGATTCTAGACG

[0031] ATCAACACAACACCACCACCAGCAACAACAACAATACCGGATGTTATTTCATGGAGAGC

[0032] AACGATGGAAGCTCTTCTGTCAAGGCTAAGATTATGGCTCATCCTCACTACCACCGTCTC

[0033] TTAGCTGCCTATGTTAATTGTCAAAAGGTAGGAGCGCCACCTGAAATGGTGGCTAGGTTA

[0034] GAGGAAGCATGCGCATCTGCCGCCACTATGGGTCCTACTAGGACCGGCTGCATAGGCGA

[0035] AGATCCTGCACTAGATCAGTTCATGGAAGCTTATTGTGAGATGCTGACTAAATACGAGCA

[0036] AGAACTCACTAAACCCTTCAAGGAAGCCATGCTTTTCCTCCAAAGGGTCGAGTGTCAGT

[0037] TCAAAGCCCTCACCGTCTCCTCTCCAAATCCTGCTTGTGGAGAGGGTGTCGACAGGAAT

[0038] GCATCATCCGAGGAAGATGTTGATGTGAACAACAATTTCATTGATCCCCTTGCAGAAGAT

[0039] CGAGAACTTAAAGGTCAGCTTCTGCGGAAGTACAGTGGATATTTAGGCAGTCTGAAGCA

[0040] GGAGTTTATGAAGAAGAGGAAGAAAGGGAAGTTGCCTAAGGAAGCCAGGCAACAGTT

[0041] GCTGGATTGGTGGAGTCGACATTACAAATGGCCTTACCCGTCGGAGTCCCAAAAGCTCG

[0042] CCTTGGCAGAGTCAACTGGTCTGGATCAGAAGCAGATAAACAACTGGTTCATTAATCAA

[0043] AGGAAAAGGCACTGGAAACCATCTGAAGATATGCAGTTTGTGGTAATGGATGCTGCCCA

[0044] TCCACACTATTATATGGACAATGTTTTGGGCAATCCCTTCCCTATGGATCTCTCTCCAACGCTTCTCTAA(SEQ ID NO.3)

[0045] The amino acid sequence encoded by GhSTM is as follows:

[0046] MEGGSSSTSCMMAFGHNSNGLCPMTMMHHPMTSHLHPQHQHQQHHHHPNSGSSSLFLPQ

[0047] PPTNNQDQNHNSSSGSSMILDDQHNTTTSNNNNTGCYFMESNDGSSSVKAKIMAHPHYHR

[0048] LLAAYVNCQKVGAPPEMVARLEEACASAATMGPTRTGCIGEDPALDQFMEAYCEMLTKYE

[0049] QELTKPFKEAMLFLQRVECQFKALTVSSPNPACGEGVDRNASSEEDVDVNNNFIDPLAEDR

[0050] ELKGQLLRKYSGYLGSLKQEFMKKRKKGKLPKEARQQLLDWWSRHYKWPYPSESQKLA

[0051] LAESTGLDQKQINNWFINQRKRHWKPSEDMQFVVMDAAHPHYYMDNVLGNPFPMDLSPTLL(SEQ IDNO.4)

[0052] The vector sequence of WMV067 is as follows:

[0053] GAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCA

[0054] CACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTA

[0055] ACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCA

[0056] GCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGCTAGAGCA

[0057] GCTTGCCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAG

[0058] TCTCAGAAGACCAAAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTC

[0059] CTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGG

[0060] TGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGC

[0061] CGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGA

[0062] CGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACA

[0063] CTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAG

[0064] ACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGT

[0065] CACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGA

[0066] TAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCC

[0067] CACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGT

[0068] GGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCA

[0069] AGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAG

[0070] TCTCTCTCTACAAATCTATCTCTCTCGAGTCTACCATGGCTCAGATCAGGTCGATGGCTCA

[0071] GGGCATTCAGACGCTGTCGCTCAACTCCTCTAATCTCTCCAAGACGCAGAAGGGGCCGC

[0072] TCGTGTCGAACTCTCTCTTCTTCGGCAGCAAGAAGCTGACACAGATCTCTGCCAAGTCA

[0073] CTGGGGGTTTTCAAGAAGGACTCGGTGCTCCGGGTGGTCCGCAAGTCCAGCTTCCGCAT

[0074] CTCAGCTTCCGTCGCTACAGCTGAGGCTGAGGCCGTGATCGCTGAGGTCTCCACTCAGC

[0075] TCAGCGAGGTTGTGGGCGTGATCGAGAGGCACCTGGAGCCAACCCTCCTGGCTGTCCAT

[0076] CTCTACGGGTCAGCGGTTGATGGTGGCCTGAAGCCCCACTCCGACATCGATCTCCTGGTT

[0077] ACAGTGACTGTCCGGCTGGACGAGACCACGAGGAGGGCTCTCATTAACGATCTCCTGG

[0078] AGACCAGCGCTTCGCCAGGCGAGTCCGAGATCCTCAGGGCGGTTGAGGTGACGATTGT

[0079] CGTTCATGACGATATCATTCCATGGAGGTACCCAGCTAAGAGGGAGCTCCAGTTCGGCG

[0080] AGTGGCAGCGCAATGACATCCTGGCCGGGATTTTCGAGCCAGCGACAATCGACATTGAT

[0081] CTGGCTATCCTCCTGACTAAGGCTAGGGAGCACTCCGTCGCTCTGGTTGGCCCTGCTGCT

[0082] GAGGAGCTCTTCGACCCAGTCCCTGAGCAGGATCTCTTCGAGGCCCTGAACGAGACCC

[0083] TCACGCTGTGGAATTCTCCGCCCGACTGGGCTGGCGATGAGAGGAATGTGGTCCTCACC

[0084] CTGTCGCGCATCTGGTACTCTGCTGTCACGGGGAAGATTGCTCCAAAGGACGTGGCTGC

[0085] TGATTGGGCGATGGAGAGGCTGCCAGCTCAGTACCAGCCTGTGATCCTCGAGGCTAGGC

[0086] AGGCTTACCTGGGCCAGGAGGAGGACAGGCTCGCGTCCCGGGCTGATCAGCTGGAGGA

[0087] GTTCGTTCATTACGTCAAGGGCGAGATTACTAAGGTCGTTGGCAAGTGACTCGAGTTTCT

[0088] CCATAATAATGTGTGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCT

[0089] CATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAA

[0090] TAAAATTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATCCCCCGAATTAATT

[0091] CGGCGTTAATTCAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCTAAGCGTCA

[0092] ATTTGTTTACACCACAATATATCCTGCCACCAGCCAGCCAACAGCTCCCCGACCGGCAGC

[0093] TCGGCACAAAATCACCACTCGATACAGGCAGCCCATCAGTCCGGGACGGCGTCAGCGG

[0094] GAGAGCCGTTGTAAGGCGGCAGACTTTGCTCATGTTACCGATGCTATTCGGAAGAACGG

[0095] CAACTAAGCTGCCGGGTTTGAAACACGGATGATCTCGCGGAGGGTAGCATGTTGATTGT

[0096] AACGATGACAGAGCGTTGCTGCCTGTGATCACCGCGGTTTCAAAATCGGCTCCGTCGAT

[0097] ACTATGTTATACGCCAACTTTGAAAACAACTTTGAAAAAGCTGTTTTCTGGTATTTAAGG

[0098] TTTTAGAATGCAAGGAACAGTGAATTGGAGTTCGTCTTGTTATAATTAGCTTCTTGGGGT

[0099] ATCTTTAAATACTGTAGAAAAGAGGAAGGAAATAATAAATGGCTAAAATGAGAATATCAC

[0100] CGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGATACGGAAGGAATGTCT

[0101] CCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAAAAATGACGGAC

[0102] AGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCT

[0103] GGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGC

[0104] AATCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACA

[0105] AAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGA

[0106] CATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTA

[0107] CTGAATAACGATCTGGCCGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAA

[0108] AGATCCGCGCGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCT

[0109] TTTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTAAGTGG

[0110] CTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACATTGCCTTCTGCG

[0111] TCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTCGAGCTATTTTTTGACTTAC

[0112] TGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTACTGGATGAATTGTTTTA

[0113] GTACCTAGAATGCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA

[0114] CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG

[0115] CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTAC

[0116] CAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTC

[0117] TAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCG

[0118] CTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGT

[0119] TGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC

[0120] GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGT

[0121] GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAA

[0122] GCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT

[0123] ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTC

[0124] GTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTG

[0125] GCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATA

[0126] ACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGC

[0127] AGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCA

[0128] TCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA

[0129] TAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGAC

[0130] ACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTAC

[0131] AGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACC

[0132] GAAACGCGCGAGGCAGGGTGCCTTGATGTGGGCGCCGGCGGTCGAGTGGCGACGGCG

[0133] CGGCTTGTCCGCGCCCTGGTAGATTGCCTGGCCGTAGGCCAGCCATTTTTGAGCGGCCA

[0134] GCGGCCGCGATAGGCCGACGCGAAGCGGCGGGGCGTAGGGAGCGCAGCGACCGAAGG

[0135] GTAGGCGCTTTTTGCAGCTCTTCGGCTGTGCGCTGGCCAGACAGTTATGCACAGGCCAG

[0136] GCGGGTTTTAAGAGTTTTAATAAGTTTTAAAGAGTTTTAGGCGGAAAAATCGCCTTTTTT

[0137] CTCTTTTATATCAGTCACTTACATGTGTGACCGGTTCCCAATGTACGGCTTTGGGTTCCCA

[0138] ATGTACGGGTTCCGGTTCCCAATGTACGGCTTTGGGTTCCCAATGTACGTGCTATCCACA

[0139] GGAAAGAGACCTTTTCGACCTTTTTCCCCTGCTAGGGCAATTTGCCCTAGCATCTGCTCC

[0140] GTACATTAGGAACCGGCGGATGCTTCGCCCTCGATCAGGTTGCGGTAGCGCATGACTAG

[0141] GATCGGGCCAGCCTGCCCCGCCTCCTCCTTCAAATCGTACTCCGGCAGGTCATTTGACCC

[0142] GATCAGCTTGCGCACGGTGAAACAGAACTTCTTGAACTCTCCGGCGCTGCCACTGCGTT

[0143] CGTAGATCGTCTTGAACAACCATCTGGCTTCTGCCTTGCCTGCGGCGCGGCGTGCCAGG

[0144] CGGTAGAGAAAACGGCCGATGCCGGGATCGATCAAAAAGTAATCGGGGTGAACCGTCA

[0145] GCACGTCCGGGTTCTTGCCTTCTGTGATCTCGCGGTACATCCAATCAGCTAGCTCGATCT

[0146] CGATGTACTCCGGCCGCCCGGTTTCGCTCTTTACGATCTTGTAGCGGCTAATCAAGGCTT

[0147] CACCCTCGGATACCGTCACCAGGCGGCCGTTCTTGGCCTTCTTCGTACGCTGCATGGCA

[0148] ACGTGCGTGGTGTTTAACCGAATGCAGGTTTCTACCAGGTCGTCTTTCTGCTTTCCGCCA

[0149] TCGGCTCGCCGGCAGAACTTGAGTACGTCCGCAACGTGTGGACGGAACACGCGGCCGG

[0150] GCTTGTCTCCCTTCCCTTCCCGGTATCGGTTCATGGATTCGGTTAGATGGGAAACCGCCA

[0151] TCAGTACCAGGTCGTAATCCCACACACTGGCCATGCCGGCCGGCCCTGCGGAAACCTCT

[0152] ACGTGCCCGTCTGGAAGCTCGTAGCGGATCACCTCGCCAGCTCGTCGGTCACGCTTCGA

[0153] CAGACGGAAAACGGCCACGTCCATGATGCTGCGACTATCGCGGGTGCCCACGTCATAGA

[0154] GCATCGGAACGAAAAAATCTGGTTGCTCGTCGCCCTTGGGCGGCTTCCTAATCGACGGC

[0155] GCACCGGCTGCCGGCGGTTGCCGGGATTCTTTGCGGATTCGATCAGCGGCCGCTTGCCA

[0156] CGATTCACCGGGGCGTGCTTCTGCCTCGATGCGTTGCCGCTGGGCGGCCTGCGCGGCCT

[0157] TCAACTTCTCCACCAGGTCATCACCCAGCGCCGCGCCGATTTGTACCGGGCCGGATGGT

[0158] TTGCGACCGTCACGCCGATTCCTCGGGCTTGGGGGTTCCAGTGCCATTGCAGGGCCGGC

[0159] AGACAACCCAGCCGCTTACGCCTGGCCAACCGCCCGTTCCTCCACACATGGGGCATTCC

[0160] ACGGCGTCGGTGCCTGGTTGTTCTTGATTTTCCATGCCGCCTCCTTTAGCCGCTAAAATT

[0161] CATCTACTCATTTATTCATTTGCTCATTTACTCTGGTAGCTGCGCGATGTATTCAGATAGCA

[0162] GCTCGGTAATGGTCTTGCCTTGGCGTACCGCGTACATCTTCAGCTTGGTGTGATCCTCCG

[0163] CCGGCAACTGAAAGTTGACCCGCTTCATGGCTGGCGTGTCTGCCAGGCTGGCCAACGTT

[0164] GCAGCCTTGCTGCTGCGTGCGCTCGGACGGCCGGCACTTAGCGTGTTTGTGCTTTTGCT

[0165] CATTTTCTCTTTACCTCATTAACTCAAATGAGTTTTGATTTAATTTCAGCGGCCAGCGCCT

[0166] GGACCTCGCGGGCAGCGTCGCCCTCGGGTTCTGATTCAAGAACGGTTGTGCCGGCGGC

[0167] GGCAGTGCCTGGGTAGCTCACGCGCTGCGTGATACGGGACTCAAGAATGGGCAGCTCGT

[0168] ACCCGGCCAGCGCCTCGGCAACCTCACCGCCGATGCGCGTGCCTTTGATCGCCCGCGAC

[0169] ACGACAAAGGCCGCTTGTAGCCTTCCATCCGTGACCTCAATGCGCTGCTTAACCAGCTC

[0170] CACCAGGTCGGCGGTGGCCCATATGTCGTAAGGGCTTGGCTGCACCGGAATCAGCACGA

[0171] AGTCGGCTGCCTTGATCGCGGACACAGCCAAGTCCGCCGCCTGGGGCGCTCCGTCGATC

[0172] ACTACGAAGTCGCGCCGGCCGATGGCCTTCACGTCGCGGTCAATCGTCGGGCGGTCGAT

[0173] GCCGACAACGGTTAGCGGTTGATCTTCCCGCACGGCCGCCCAATCGCGGGCACTGCCCT

[0174] GGGGATCGGAATCGACTAACAGAACATCGGCCCCGGCGAGTTGCAGGGCGCGGGCTAG

[0175] ATGGGTTGCGATGGTCGTCTTGCCTGACCCGCCTTTCTGGTTAAGTACAGCGATAACCTT

[0176] CATGCGTTCCCCTTGCGTATTTGTTTATTTACTCATCGCATCATATACGCAGCGACCGCATG

[0177] ACGCAAGCTGTTTTACTCAAATACACATCACCTTTTTAGACGGCGGCGCTCGGTTTCTTC

[0178] AGCGGCCAAGCTGGCCGGCCAGGCCGCCAGCTTGGCATCAGACAAACCGGCCAGGATT

[0179] TCATGCAGCCGCACGGTTGAGACGTGCGCGGGCGGCTCGAACACGTACCCGGCCGCGA

[0180] TCATCTCCGCCTCGATCTCTTCGGTAATGAAAAACGGTTCGTCCTGGCCGTCCTGGTGCG

[0181] GTTTCATGCTTGTTCCTCTTGGCGTTCATTCTCGGCGGCCGCCAGGGCGTCGGCCTCGGT

[0182] CAATGCGTCCTCACGGAAGGCACCGCGCCGCCTGGCCTCGGTGGGCGTCACTTCCTCGC

[0183] TGCGCTCAAGTGCGCGGTACAGGGTCGAGCGATGCACGCCAAGCAGTGCAGCCGCCTC

[0184] TTTCACGGTGCGGCCTTCCTGGTCGATCAGCTCGCGGGCGTGCGCGATCTGTGCCGGGG

[0185] TGAGGGTAGGGCGGGGGCCAAACTTCACGCCTCGGGCCTTGGCGGCCTCGCGCCCGCT

[0186] CCGGGTGCGGTCGATGATTAGGGAACGCTCGAACTCGGCAATGCCGGCGAACACGGTC

[0187] AACACCATGCGGCCGGCCGGCGTGGTGGTGTCGGCCCACGGCTCTGCCAGGCTACGCA

[0188] GGCCCGCGCCGGCCTCCTGGATGCGCTCGGCAATGTCCAGTAGGTCGCGGGTGCTGCGG

[0189] GCCAGGCGGTCTAGCCTGGTCACTGTCACAACGTCGCCAGGGCGTAGGTGGTCAAGCA

[0190] TCCTGGCCAGCTCCGGGCGGTCGCGCCTGGTGCCGGTGATCTTCTCGGAAAACAGCTTG

[0191] GTGCAGCCGGCCGCGTGCAGTTCGGCCCGTTGGTTGGTCAAGTCCTGGTCGTCGGTGCT

[0192] GACGCGGGCATAGCCCAGCAGGCCAGCGGCGGCGCTCTTGTTCATGGCGTAATGTCTCC

[0193] GGTTCTAGTCGCAAGTATTCTACTTTATGCGACTAAAACACGCGACAAGAAAACGCCAG

[0194] GAAAAGGGCAGGGCGGCAGCCTGTCGCGTAACTTAGGACTTGTGCGACATGTCGTTTTC

[0195] AGAAGACGGCTGCACTGAACGTCAGAAGCCGACTGCACTATAGCAGCGGAGGGGTTGG

[0196] ATCAAAGTACTTTGATCCCGAGGGGAACCCTGTGGTTGGCATGCACATACAAATGGACG

[0197] AACGGATAAACCTTTTCACGCCCTTTTAAATATCCGTTATTCTAATAAACGCTCTTTTCTC

[0198] TTAGGTTTACCCGCCAATATATCCTGTCAAACACTGATAGTTTAAACTGAAGGCGGGAAA

[0199] CGACAATCTGATCCAAGCTCAAGCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTT

[0200] GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATG

[0201] TGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAA

[0202] CGACGGCCAGTGCCAAGCTAAGCTTGCATGCCTGCAGGTCCCCAGATTAGCCTTTTCAA

[0203] TTTCAGAAAGAATGCTAACCCACAGATGGTTAGAGAGGCTTACGCAGCAGGTCTCATCA

[0204] AGACGATCTACCCGAGCAATAATCTCCAGGAAATCAAATACCTTCCCAAGAAGGTTAAA

[0205] GATGCAGTCAAAGATTCAGGACTAACTGCATCAAGAACACAGAAAGATATATTCT

[0206] CAAGATCAGAAGTACTATTCCAGTATGGACGATTCAAGGCTTGCTTCACAAACCAAGGC

[0207] AAGTAATAGAGATTGGAGTCTCTAAAAAGGTAGTTCCACTGAATCAAAGGCCATGGAG

[0208] TCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCAT

[0209] ACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACG

[0210] ACACACTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATT

[0211] GAGACTTTTCAACAAAGGGTAATATCCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATC

[0212] TGTCACTTTATTGTGAAGATAGTGGAAAGGAAGGTGGCTCCTACAAATGCCATCATTGC

[0213] GATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGAC

[0214] CCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCA

[0215] AGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTC

[0216] GCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGAACACGGGGGACgtcg

[0217] acTCTAGAGGATCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATC

[0218] CTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCG

[0219] AGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT

[0220] GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCC

[0221] GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC

[0222] GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG

[0223] TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA

[0224] GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTC

[0225] TATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAA

[0226] CATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGC

[0227] GACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA

[0228] AAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGG

[0229] GATCACTCTCGGCATGGACGAGCTGTACAAGTGAACTAGTGAGCTCGAATTTCCCCGAT

[0230] CGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATG

[0231] ATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGAC

[0232] GTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAG

[0233] AAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGG(SEQ ID NO.5)

[0234] Example 2: Agrobacterium-mediated genetic transformation of cotton

[0235] The cotton was transformed by Agrobacterium-mediated method. The detailed steps and methods are as follows: Xiaoyang, Ge, Jieting, Xu, Zhaoen, Yang et al. Efficient genotype-independent cotton genetic transformation and genome editing. [J]. J Integr Plant Biol, 2022, 65: 0.

[0236] 1) Before infection, the above-mentioned Agrobacterium strain containing WMV067-GhSTM and the Agrobacterium strain containing WMV067 empty vector were inoculated on YEP solid medium containing 100 mg / mL kanamycin and 100 mg / mL rifampicin, and resuscitated and cultured at 28°C in the dark for 2 days. Pick a single colony for activation and shake culture overnight (28°C) to obtain an activated bacterial solution, and then shake WMV067-GhSTM / Agrobacterium and WMV067 / Agrobacterium to OD 600 = between 0.6 and 0.9, for standby use.

[0237] 2) One day before infection, 200 delinted and uncoated cotton seeds (Yuanmian No. 8, Xinhai 78 and Shixiya No. 1) were disinfected with mercuric chloride and rinsed with sterile water for 4 times, added to MSB culture medium, and cultured at 28°C overnight for 24 hours.

[0238] 3) Select healthy and germinating seeds for transformation, hold the seeds with tweezers, peel the seed coat with a scalpel, and expose the stem tip. Place the peeled stem tip (explant) into the WMV067-GhSTM / Agrobacterium and WMV067 / Agrobacterium culture solution with adjusted OD value, put it into an ultrasonic cleaner for ultrasonic treatment for 40 seconds, and then put it into a greenhouse shaker for 50 minutes (25°C) for infection. After infection, place the explant in sterile filter paper and blow dry, place it in the co-culture medium, and culture it in the dark at 23°C.

[0239] 4) After 3-4 days of co-cultivation, the explants are transferred to a recovery medium and cultured for 4 days, and then transferred to a screening medium for bud induction and positive seedling screening, and bud elongation is induced in a bud elongation medium; finally, the positive seedlings are induced to take root and transplanted into pots, and the resulting plants are T0 generation transgenic plants.

[0240] 5) PCR detection: Extract genomic DNA from leaves of T0 transgenic plants and use it as a template. AADA-specific primers (AADA-F1 / R1) are used for amplification and agarose gel electrophoresis is performed. Positive plants can be specifically amplified and detected (e.g. Figure 2 As shown in Figure 2 ). The PCR amplification product has a target band of 435 bp, which can be determined as a positive plant. The positive plants detected by the above experiment are used for later transplantation.

[0241] AADA-F1: 5'-CAGGGTGAGGACCACATTCC-3' (SEQ ID NO. 6);

[0242] AADA-R1: 5'-TCCGACATCGATCTCCTGGT-3' (SEQ ID NO.7)

[0243] In the present invention, the formula of the YEP solid culture medium is: 10g beef extract, 10g yeast extract, 10g NaCl, 15g agar powder, water is added to make the volume to 1L, the pH value is adjusted to 7.0, and high-pressure sterilization is carried out at 121°C for 20min.

[0244] The composition of the co-culture medium is: 10 ml / L CA mother solution, 30 g / L glucose, 4.2 g / L MES (morpholineethanesulfonic acid), 0.1 ml / L B5 vitamins (G219), 1 mg / L 6-BA (6-benzyladenine), 0.1 mg / L NAA (naphthaleneacetic acid), 0.2 mM acetosyringone, 200 mg / mL cysteine ​​(CYS), pH 5.4.

[0245] The composition of the recovery medium (R0) is: 4.4g / L MS salt and B5 vitamin mixture (model M404), 20g / L glucose, 1.29g / L calcium gluconate, 4g / L agar, 1mg / L 6-BA, 0.1mg / L NAA, 100mg / L carbenicillin, 100mg / L cephalosporin, pH 5.6.

[0246] The composition of the screening medium is: 4.4g / L MS salt and B5 vitamin mixture (model M404), 20g / L glucose, 1.29g / L calcium gluconate, 4g / L agar, 1mg / L 6-BA, 0.1mg / L NAA, 100mg / L carbenicillin, 100mg / L cephalosporin, 100mg / L spectinomycin, pH 5.6.

[0247] The composition of the shoot elongation medium is: 4.4 g / L MS salt and B5 vitamin mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / L spectinomycin, pH 5.6.

[0248] Example 3: GhSTM overexpression improves transformation efficiency

[0249] The results are shown in Table 1. Compared with the empty vector WMV067, the treatment of WMV067-GhSTM vector can greatly improve the genetic transformation efficiency of the same variety. Among them, the transformation efficiency of Yuanmian 8 as the receptor transformation empty vector WMV067 is 1.20-1.82%, while the vector overexpressing GhSTM can increase the transformation efficiency to 7.14-7.92%; the transformation efficiency of Xinhai 78 as the receptor transformation empty vector control is 0.50-0.95%, while the vector overexpressing GhSTM can increase the transformation efficiency to 1.08-2.50%, and the Asian cotton stone system Asia 1 has increased from the original 0.91-1.30% to 2.27-2.61%. Figure 3 The budding conditions of GhSTM gene-transfected materials and empty vector-transfected control materials are shown in Figure 2. Figure 3 It can be seen that compared with the empty vector control transformed with WMV067, overexpression of GhSTM can screen out more green shoots, improve regeneration efficiency and shorten transformation time.

[0250] Transformation efficiency = (positive plants / number of shoot tips) × 100%

[0251] It can be seen that Agrobacterium overexpressing the GhSTM gene can greatly improve the transformation efficiency of cotton, especially solving the genotype restriction problem of some major cotton varieties with low transformation efficiency.

[0252] Table 1

[0253]

Claims

1. Application of GhSTM gene in improving the efficiency of cotton genetic transformation.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the GhSTM gene is shown in SEQ ID NO.

3.

3. The use according to claim 2, characterized in that: The GhSTM gene was amplified using the 49 genome of the China Cotton Research Institute as a template through the primers shown in SEQ ID NO.1 and SEQ ID NO.

2.

4. The use according to claim 1, characterized in that: The amino acid sequence encoded by the GhSTM gene is shown in SEQ ID NO.

4.

5. The use according to claim 1, characterized in that: The GhSTM gene was constructed onto the cotton vector WMV067 and transformed into cotton, thereby enhancing the expression of the GhSTM gene in the cotton material and improving the efficiency of cotton genetic transformation.

6. Recombinant expression vector and genetically engineered bacteria containing the GhSTM gene.

7. Use of the recombinant expression vector and genetically engineered bacteria according to claim 6 in improving the genetic transformation efficiency of cotton.

8. Protein encoded by GhSTM gene.

9. Application of the protein encoded by the GhSTM gene in improving the efficiency of cotton genetic transformation.

10. Application of GhSTM gene in cotton breeding.

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