Application of GhSTM gene in improving genetic transformation efficiency of cotton

Abstract

The application discloses application of a GhSTM gene in improving cotton genetic transformation efficiency.A novel GhSTM gene is cloned from cotton, a DNA sequence of a full-length CDS fragment of the gene is constructed into an overexpression vector, then the vector is introduced into Agrobacterium, and cotton is transformed by using an Agrobacterium-mediated method, and it is found that, compared with a WMV067 empty vector control, more green sprouts can be screened out by overexpressing the GhSTM, the regeneration efficiency is improved, and the transformation time is shortened.Meanwhile, the Agrobacterium overexpressing the GhSTM gene can greatly improve the transformation efficiency on cotton, and especially, the genotype limitation problem of some cotton varieties with low transformation efficiency is solved.Therefore, overexpressing the gene and applying the gene to commercial cotton varieties with difficult genetic transformation can help to improve the genetic transformation efficiency of the cotton varieties, and further improve the yield, quality and stress resistance of high-quality cotton germplasm, and the application has important economic value and social benefits.

Description

Technical Field

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

[0002] Cotton is one of the world's most important cash crops. my country is both the world's largest cotton producer and consumer. The development of cotton production is closely linked to the selection and promotion of new varieties. Currently promoted varieties or the direction of variety selection 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 foundation and core of plant genetic engineering. Researchers have attempted to improve cotton using various transformation methods, such as gene gun-mediated, Agrobacterium-mediated, and pollen tube-mediated, resulting in the development of new insect-resistant and herbicide-resistant cotton varieties and materials. Genetically modified cotton not only increases agricultural yield, improves quality, and increases farmers' income, but also reduces pesticide use, protecting the environment and biodiversity. However, cotton is a crop that is relatively difficult to genetically transform. Global research and industrialization of genetically modified cotton lags significantly. Inefficient genetic transformation technology is one of the bottlenecks limiting the development of genetically modified cotton.

[0003] The genetic transformation efficiency of cotton has always been low, but significant 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. This method breaks the variety limitations of traditional transgenic methods and can be used to genetically modify any cotton variety, quickly obtaining transgenic cotton materials. To a certain extent, it breaks the genotype dependence of cotton transformation. Despite this, cotton's genotype dependence is still very serious. The transformation efficiency of some commercial varieties such as Yuanmian No. 8, Xinlu Zao series, Tahe No. 2, as well as 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 cotton varieties, 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] While numerous tissue culture protocols and technologies have achieved some success in improving cotton genetic transformation, they clearly cannot resolve the issue of genotype limitations. Plant tissue culture regeneration, a key trait influencing the success of transgenic technology, is inevitably controlled by one or more major genes. Therefore, cloning and utilizing regeneration-related genes, screening for key regeneration factors, and developing broadly applicable molecular tools that enhance crop regeneration and enable genotype-independent genetic transformation are key to addressing the genotype dependency of cotton genetic transformation. Summary of the Invention

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

[0006] In order to achieve the above objectives, the technical solution of the present invention is, on the one hand, to provide the application of the 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 using 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 into 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.

[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 the GhSTM gene in cotton breeding.

[0016] Beneficial effects of the present invention:

[0017] The present invention cloned a novel GhSTM gene from cotton and verified its function of improving genetic transformation efficiency in cotton. The present invention constructed an overexpression vector with the DNA sequence of the full-length CDS fragment of the gene, which was then introduced into Agrobacterium and transformed into cotton using the Agrobacterium-mediated method. In this process, it was found that compared with the empty vector control WMV067, overexpression of GhSTM can screen out more green buds, improve regeneration efficiency, increase the efficiency of obtaining positive plants from the stem apical meristem, and shorten the transformation time. Therefore, overexpressing this gene and applying it 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 use of the GhSTM gene in the genetic transformation of upland cotton. The invention constructs a WMV067-GhSTM overexpression vector and uses Agrobacterium-mediated transformation to transform the Xinjiang main cultivar Yuanmian No. 8, which suffers from severe genotypic restrictions. The transformation efficiency is increased from 1.20-1.82% to 7.14-7.92%. The transformation efficiency of the Sea Island cotton variety Xinhai 78 is increased from 0.50-0.95% to 1.08-2.50%, and the transformation efficiency of the Asian cotton stone line Ya No. 1 is increased from 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 the 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 marker III molecular weight standard.

[0021] Figure 3 This is the budding situation of GhSTM gene-transfected materials and empty vector-transfected control materials. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are further described in detail below with reference to the examples.

[0023] Example 1: GhSTM gene cloning

[0024] According to the sequence of the cotton GhSTM gene (>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 high-efficiency transgenic receptor 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 1128bp fragment, i.e., the GhSTM gene with restriction enzyme cutting site (the sequence of GhSTM gene 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 ligation reaction, and the ligation product was transformed into Escherichia coli competent cells. After resistance screening, single clones were selected for sequencing, and the correctly sequenced vector was named WMV067-GhSTM (structure is shown in Figure 1 WMV067-GhSTM was transformed into competent Agrobacterium EHA105, and positive clones were selected for use. Simultaneously, the WMV067 (WMV067-GFP) empty vector was transformed into competent Agrobacterium 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 using Agrobacterium-mediated transformation. 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 the WMV067 empty vector were inoculated onto YEP solid medium containing 100 mg / mL kanamycin and 100 mg / mL rifampicin, and cultured for 2 days in the dark at 28°C. Single colonies were picked and activated overnight with shaking culture (28°C) to obtain activated bacterial solution, and then the WMV067-GhSTM / Agrobacterium and WMV067 / Agrobacterium were shaken to OD 600 = between 0.6-0.9, 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 four times with sterile water. They were then added to MSB culture medium and cultured overnight at 28°C for 24 h.

[0238] 3) Select healthy, germinating seeds for transformation. Grasp the seeds with tweezers and peel the seed coat with a scalpel to expose the stem apex. Place the peeled stem apex (explant) into a WMV067-GhSTM / Agrobacterium and WMV067 / Agrobacterium culture solution adjusted to the appropriate OD value. Ultrasonicate in an ultrasonic cleaner for 40 seconds. Then, shake in a greenhouse shaker for 50 minutes (25°C) for infection. After infection, place the explant on sterile filter paper and air-dry. Place in a co-culture medium and incubate at 23°C in the dark.

[0239] 4) After 3-4 days of co-cultivation, the explants are transferred to a recovery medium and cultured for 4 days. Thereafter, the explants are 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 root and transplanted into pots to obtain T0 generation transgenic plants.

[0240] 5) PCR detection: Genomic DNA of leaves of T0 transgenic plants was extracted and used as template to amplify using AADA specific primers (AADA-F1 / R1) and then subjected to agarose gel electrophoresis. Positive plants (such as Figure 2 As shown in Figure 2 ). The PCR amplification product has a 435 bp target band, which can be determined as a positive plant. Positive plants detected through the above experiment are used for later transplanting.

[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, after adding water to make the volume to 1L, the pH value is adjusted to 7.0, and autoclaved 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 / LB5 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: 4.4 g / L MS salts and B5 vitamin mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, pH 5.6.

[0246] The screening medium consists of 4.4 g / L MS salts and B5 vitamin mixture (model M404), 20 g / L glucose, 1.29 g / L calcium gluconate, 4 g / L agar, 1 mg / L 6-BA, 0.1 mg / L NAA, 100 mg / L carbenicillin, 100 mg / L cephalosporin, 100 mg / 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, shown in Table 1, show that treatment with the WMV067-GhSTM vector significantly improved the genetic transformation efficiency of the same varieties compared to the empty vector WMV067. Specifically, when Yuanmian 8 was used as the recipient, the empty vector WMV067 had a transformation efficiency of 1.20-1.82%, while the vector overexpressing GhSTM increased the transformation efficiency to 7.14-7.92%. When Xinhai 78 was used as the recipient, the empty vector control had a transformation efficiency of 0.50-0.95%, while the vector overexpressing GhSTM increased the transformation efficiency to 1.08-2.50%. Furthermore, the transformation efficiency of the Asian cotton stone strain Ya No. 1 increased from 0.91-1.30% to 2.27-2.61%. Figure 3 The budding situation of GhSTM gene-transfected materials and empty vector-transfected control materials. 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 stem tips) × 100%

[0251] It can be seen from this 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 overexpression of GhSTM gene in improving cotton genetic transformation efficiency; the nucleotide sequence of the GhSTM gene is shown in SEQ ID NO.

3.

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

2.

3. 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.

4. The use according to claim 1, characterized in that The GhSTM gene was constructed into 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.

5. Application of a recombinant expression vector and genetically engineered bacteria containing the GhSTM gene in improving the efficiency of genetic transformation of cotton; the nucleotide sequence of the GhSTM gene is shown in SEQ ID NO.3.

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