CN115786365B - Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application - Google Patents
Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application Download PDFInfo
- Publication number
- CN115786365B CN115786365B CN202211311985.0A CN202211311985A CN115786365B CN 115786365 B CN115786365 B CN 115786365B CN 202211311985 A CN202211311985 A CN 202211311985A CN 115786365 B CN115786365 B CN 115786365B
- Authority
- CN
- China
- Prior art keywords
- ghgt
- gene
- cotton
- verticillium wilt
- application
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 75
- 229920000742 Cotton Polymers 0.000 title claims abstract description 54
- 230000009261 transgenic effect Effects 0.000 title abstract description 37
- 239000013604 expression vector Substances 0.000 title abstract description 16
- 238000000034 method Methods 0.000 title abstract description 12
- 102000004169 proteins and genes Human genes 0.000 title abstract description 11
- 241000082085 Verticillium <Phyllachorales> Species 0.000 claims abstract description 30
- 241000219195 Arabidopsis thaliana Species 0.000 claims abstract description 16
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 230000003803 hair density Effects 0.000 claims description 8
- 241000196324 Embryophyta Species 0.000 abstract description 46
- 210000004209 hair Anatomy 0.000 abstract description 12
- 230000001105 regulatory effect Effects 0.000 abstract description 4
- 235000013399 edible fruits Nutrition 0.000 abstract description 3
- 239000000463 material Substances 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 244000299507 Gossypium hirsutum Species 0.000 description 48
- 239000013598 vector Substances 0.000 description 28
- 238000006243 chemical reaction Methods 0.000 description 17
- 241000219194 Arabidopsis Species 0.000 description 16
- 230000002018 overexpression Effects 0.000 description 13
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 12
- 230000014509 gene expression Effects 0.000 description 10
- 241000589158 Agrobacterium Species 0.000 description 9
- 238000010367 cloning Methods 0.000 description 9
- 241000707216 Scopelarchidae Species 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 108091023040 Transcription factor Proteins 0.000 description 7
- 102000040945 Transcription factor Human genes 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 210000002615 epidermis Anatomy 0.000 description 6
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 6
- 229960004889 salicylic acid Drugs 0.000 description 6
- 208000035240 Disease Resistance Diseases 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 235000009429 Gossypium barbadense Nutrition 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 235000018322 upland cotton Nutrition 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 description 1
- 101100327005 Arabidopsis thaliana CBP60G gene Proteins 0.000 description 1
- 101100382172 Arabidopsis thaliana CYP71A13 gene Proteins 0.000 description 1
- 101100011910 Arabidopsis thaliana EARLI1 gene Proteins 0.000 description 1
- 101100333151 Arabidopsis thaliana EGC2 gene Proteins 0.000 description 1
- 101100448366 Arabidopsis thaliana GH3.12 gene Proteins 0.000 description 1
- 101100449814 Arabidopsis thaliana GTL1 gene Proteins 0.000 description 1
- 101100533570 Arabidopsis thaliana SIRK gene Proteins 0.000 description 1
- 101150023103 CHI gene Proteins 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 101150089682 FRK1 gene Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101000926140 Homo sapiens Gem-associated protein 2 Proteins 0.000 description 1
- 101000716750 Homo sapiens Protein SCAF11 Proteins 0.000 description 1
- 101000723833 Homo sapiens Zinc finger E-box-binding homeobox 2 Proteins 0.000 description 1
- 101150049628 MPK4 gene Proteins 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101000742142 Phaseolus vulgaris Pathogenesis-related protein 1 Proteins 0.000 description 1
- 102100020876 Protein SCAF11 Human genes 0.000 description 1
- 241000589615 Pseudomonas syringae Species 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 244000194806 Solanum sisymbriifolium Species 0.000 description 1
- 235000018724 Solanum sisymbriifolium Nutrition 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000024346 drought recovery Effects 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000009746 freeze damage Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 230000021918 systemic acquired resistance Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 108091005957 yellow fluorescent proteins Proteins 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明提供了一种棉花GhGT‑3b_A04基因、蛋白质、表达载体、转基因植物获取方法和应用，属于分子生物学和生物技术领域，所述棉花GhGT‑3b_A04基因的核苷酸序列如SEQ ID No.1所示。GhGT‑3b_A04基因能增强拟南芥黄萎病抗性，且获得的转基因植株均能正常开花结果，转化基因能稳定遗传至下一代，故该基因可以进一步运用于黄萎病抗性改良，培育出抗黄萎病新品种，为植物黄萎病抗性改良提供新途径；本发明为植物根系和表皮毛发育调控提供理论依据；本发明为黄萎病抗性改良、植物根系和表皮毛研究提供新材料。
The present invention provides a cotton GhGT‑3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application, belonging to the fields of molecular biology and biotechnology, and the nucleotide sequence of the cotton GhGT‑3b_A04 gene is shown in SEQ ID No. 1. The GhGT‑3b_A04 gene can enhance the resistance of Arabidopsis thaliana to Verticillium wilt, and the obtained transgenic plants can bloom and bear fruit normally, and the transformed gene can be stably inherited to the next generation, so the gene can be further used for improving resistance to Verticillium wilt, cultivating new varieties resistant to Verticillium wilt, and providing a new way for improving resistance to Verticillium wilt in plants; the present invention provides a theoretical basis for regulating the development of plant roots and epidermal hairs; the present invention provides new materials for improving resistance to Verticillium wilt and studying plant roots and epidermal hairs.
Description
Technical Field
The invention belongs to the field of molecular biology and biotechnology, and particularly relates to a cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application.
Background
China is a large country for cotton production and consumption, and cotton has important roles in national economy in China. Verticillium wilt is a soil-borne fungal disease that severely affects cotton yield and quality. An effective way for preventing and treating verticillium wilt is to cultivate a cultivated disease-resistant variety, so that the identification of related genes against verticillium wilt, the exploration of disease resistance mechanism thereof, and the key of cotton disease resistance molecular breeding are provided. Salicylic acid is an important defensive hormone, having an important role in the plant's defense response against pathogens and in the establishment of systemic acquired resistance. In cotton, the salicylic acid signaling pathway has been shown to be involved in the disease-resistant process of verticillium wilt.
The DNA binding domain of triple helix Trihelix transcription factor, also known as GT transcription factor, contains 3 consecutive alpha helices, which can specifically bind to the GT cis element (GT 1 box,5'-GGTTAA-3'; GT2 box,5'-GGTAAT-3'; GT3 box,5 '-GGTAAA-3'). GT transcription factors can be classified into three classes, GT-1, GT-2 and GT-3, depending on the number of domains. The five subfamilies of GT-1, GT-2, GT gamma, SH4 and SIP1 can be distinguished according to alpha helical domain changes. Trihelix transcription factors are involved in regulating plant biotic and abiotic stress as demonstrated by studies of the function of some triple helix Trihelix transcription factors. The overexpression of the genes of the rice OsGT gamma-1, the OsGT gamma-2 and the OsGT gamma-3 can improve the salt tolerance of plants. The plants of the soybean GmGT-2a and GmGT-2b gene heterologous expression have improved tolerance to salt, freeze injury and drought stress. The ShCIGT gene over-expression tomato plant in the wild tomato has higher cold tolerance. The density and the number of the air holes of the arabidopsis gtl1 mutant are obviously reduced, and the drought tolerance and the water utilization rate are improved. GTL1 interacts with MPK4, directly binds and modulates salicylic acid biosynthesis, transport and response related genes, upregulates defensive responses to pseudomonas syringae strains and inhibits factors that regulate growth and development. In cotton, 102 Trihelix transcription factors were identified in the upland cotton genome, wherein Gh_A05G2067 (GT-2) was up-regulated under drought and salt stress conditions, which could increase cotton tolerance to drought and salt stress. However, the biological functions of other cotton Trihelix transcription factors, particularly those that regulate the disease resistance response of cotton through salicylic acid synthesis and signal transduction pathways, are not known.
Disclosure of Invention
In view of the above, the invention aims to provide a cotton GhGT-3b_A04 gene, a protein, an expression vector, a transgenic plant acquisition method and application, wherein the cotton GhGT-3b_A04 gene can improve verticillium wilt resistance of plants.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a cotton GhGT-3b_A04 gene, and the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown as SEQ ID No. 1.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving verticillium wilt resistance of plants.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving the plant root hair length.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving plant root hair density.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving the plant epidermis hair density.
The invention also provides a protein coded by the cotton GhGT-3b_A04 gene, and the amino acid sequence of the protein is shown as SEQ ID No. 2.
The invention also provides an over-expression vector which contains the cotton GhGT-3b_A04 gene according to the technical scheme.
Preferably, the preparation method of the over-expression vector comprises the following steps:
1) Performing BP reaction on the cotton GhGT-3b_A04 gene and an entry vector pDONR-Zeo to obtain a pDONR-Zeo-GhGT-3b_A04 entry vector;
2) And 4) carrying out LR reaction on the pDONR-Zeo-GhGT-3b_A04 entry vector obtained in the step 1) and a target vector pEarley gate 101 to obtain an over-expression vector.
The invention also provides a method for obtaining the transgenic plant, which comprises the following steps:
a. the over-expression vector in the technical scheme is transformed into agrobacterium to obtain transformed bacteria;
b. and c, infecting plants with the transformed bacteria obtained in the step a to obtain transgenic plants.
Preferably, the agrobacterium comprises agrobacterium GV3101.
The beneficial effects of the invention are as follows:
1. The GhGT-3b_A04 gene can enhance verticillium wilt resistance of arabidopsis, the obtained transgenic plants can normally bloom and fruit, and the transformed genes can be stably inherited to the next generation, so that the gene can be further applied to verticillium wilt resistance improvement, a new variety resisting verticillium wilt is cultivated, and a new way is provided for plant verticillium wilt resistance improvement;
2. the invention provides a theoretical basis for regulating and controlling plant root system and epidermis hair growth.
3. The invention provides a new material for the verticillium wilt resistance improvement and the research of plant root systems and epidermal hairs.
Drawings
FIG. 1 is a schematic diagram showing the gene expression of cotton GhGT-3b_A04 gene sequence, cloning and application thereof;
FIG. 2 is a schematic diagram showing the identification of Arabidopsis plants and plant phenotypes of the cotton GhGT-3b_A04 gene sequence, cloning and application thereof;
FIG. 3 is a schematic diagram showing the gene sequence, cloning and application subcellular localization of cotton GhGT-3b_A04 according to the present invention;
FIG. 4 is a schematic diagram showing the gene sequence, cloning and application of GhGT-3b_A04 and the treatment of verticillium wilt of Arabidopsis thaliana;
FIG. 5 is a schematic diagram showing DAB staining after verticillium wilt treatment of cotton GhGT-3b_A04 gene sequence, cloning and application thereof;
FIG. 6 is a schematic diagram showing the growth of root system of transgenic Arabidopsis thaliana seedlings, in which the cotton GhGT-3b_A04 gene sequence and cloning and application thereof of the present invention are shown;
FIG. 7 is a schematic diagram of cotton GhGT-3b_A04 gene sequence, cloning and application of the gene Arabidopsis rosette leaf epidermis hair phenotype;
FIG. 8 is a diagram showing the expression level change of the gene GhGT-3b_A04 gene sequence of cotton, cloning and application of the gene and the Arabidopsis rosette She Kangbing.
Detailed Description
The invention provides a cotton GhGT-3b_A04 gene, wherein the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown as SEQ ID No.1, and the specific steps are as follows:
ATGGAGGGACATCATCGCCATCATCATCATCAGCAGCAGCAACATCTGCAGCAACAACAACAGCCTGTAAGTGTCAACGTTGAAGCTGATAGGTTTCCTCAATGGAGTGTTCAAGAGACAAAGGATTTTTTAATGATCAGAGCAGAGCTGGATCAAAGTTTCATGGAGACCAAAAGGAACAAGCTGCTTTGGGAAGTTATCTCCACCAGGATGAGGGAAAAGGGTTATAATCGAAGCGCTGAACAGTGCAAGTGCAAGTGGAAAAACCTCTTTACTCGTTACAAGGGATGTGGAACGGTTGACGCAGAAATTGTGCGGCAACAGTTCCCGTTTTACGACGAGTTGCAGGCCATATTCACGGCGAGGATGCAAAGTGTTCTATGTTCGGAAAGTGAAGGCGGAGGAGCTACGGGGTCGAAAAAAAAGGCAGCACAGGCGCAGCTATCTTCCGACGAGGAAGACGATACGGAGGAAAACGAGTACAGCTTTAGGAAGAAGAAGAAAGGGAAAACTGGTGGTGGGAGTAGTAGTACGAGCGGCAGCATTAATATAAAGGAAATGTTAGCGAACTTCATGAGGCAACAGTTGCAGATGGAAATGCAATGGAGGGAAGCTTTGGAGTCGAGAGAAAACGAGAGGCGGATGAAGGAAATGGAGTGGAGGCAAACCATGGAAGCCCTTGAAAACGAGAGGATAATGATGGAGAGAAGGTGGAGGGAAAGGGAAGAACAAAGGAGAATAAGGGAAGAAGCTAGGGCTGAGAAGAGAGATGCTCTCATTACTGCACTTCTAAACAAGCTTAGAAGAGAA GATCAAATGTAG.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving verticillium wilt resistance of plants.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving the plant root hair length.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving plant root hair density.
The invention also provides application of the cotton GhGT-3b_A04 gene in improving the plant epidermis hair density.
In the present invention, the plant preferably comprises arabidopsis thaliana.
The HIA of the invention provides the protein encoded by the cotton GhGT-3b_A04 gene according to the technical scheme, and is characterized in that the amino acid sequence of the protein is shown as SEQ ID No.2, and the protein is specifically as follows:
MEGHHRHHHHQQQQHLQQQQQPVSVNVEADRFPQWSVQETKDFLMIRAELDQSFMETKRNKLLWEVISTRMREKGYNRSAEQCKCKWKNLFTRYKGCGTVDAEIVRQQFPFYDELQAIFTARMQSVLCSESEGGGATGSKKKAAQAQLSSDEEDDTEENEYSFRKKKKGKTGGGSSSTSGSINIKEMLANFMRQQLQMEMQWREALESRENERRMKEMEWRQTMEALENERIMMERRWREREEQRRIREEARAEKRDALI TALLNKLRRE DQM.
The invention also provides an over-expression vector, which contains the cotton GhGT-3b_A04 gene of claim 1.
In the present invention, the method for preparing the overexpression vector preferably comprises the following steps:
1) Performing BP reaction on the cotton GhGT-3b_A04 gene and an entry vector pDONR-Zeo to obtain a pDONR-Zeo-GhGT-3b_A04 entry vector;
2) And 4) carrying out LR reaction on the pDONR-Zeo-GhGT-3b_A04 entry vector obtained in the step 1) and a target vector pEarley gate 101 to obtain an over-expression vector.
The cotton GhGT-3b_A04 gene and the entry vector pDONR-Zeo are subjected to BP reaction to obtain the pDONR-Zeo-GhGT-3b_A04 entry vector. In the present invention, the entry vector pDONR-Zeo is preferably purchased from Invitrogen corporation. In the present invention, the conditions for the BP reaction preferably include 1. Mu.l of the reaction system for the pDONR-Zeo vector, 1. Mu.l of the PCR product, 0.5. Mu.l of BP reaction enzyme (Invitrogen, california, USA), and 25℃for 2 hours.
The present invention preferably provides an overexpression vector by LR reaction of the pDONR-Zeo-GhGT-3b_A04 entry vector with the target vector pEarley Gate 101. In the present invention, the objective vector pEarley Gate 101 is preferably purchased from Ubbelopsis. In the present invention, the conditions for the LR reaction preferably include 1. Mu.l of the reaction system pDONR-Zeo-GhGT-3b_A04 vector, 1. Mu.l of PCR product, 0.5. Mu.l of LR-reactive enzyme (Invitrogen, california, U.S.A.), and 25℃for 1 hour.
The invention also provides a method for obtaining the transgenic plant, which comprises the following steps:
a. the over-expression vector in the technical scheme is transformed into agrobacterium to obtain transformed bacteria;
b. and c, infecting plants with the transformed bacteria obtained in the step a to obtain transgenic plants.
The over-expression vector in the technical scheme is transformed into agrobacterium to obtain transformed bacteria. In the present invention, the agrobacterium preferably includes agrobacterium GV3101. The method of transformation is not particularly limited, and those skilled in the art can operate conventionally.
The transformed bacteria obtained in the step a are infected to plants to obtain transgenic plants. The method of the present invention is not particularly limited, and those skilled in the art can use conventional operations, and will not be described here.
In the present invention, the plant preferably comprises arabidopsis thaliana. In the invention, the GhGT-3b_A04 gene can enhance the verticillium wilt resistance of arabidopsis, the obtained transgenic plants can normally bloom and fruit, and the transformed genes can be stably inherited to the next generation.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Cloning and expression vector construction of cotton GhGT-3b_A04 gene
(1) Primer design
According to cottonFGD reported cotton genome sequences, the GhGT-3b_A04 gene cDNA sequence (SEQ ID No. 1) is determined through bioinformatics comparison, and a primer of the full-length sequence of a coding frame (ORF) is designed according to the sequence, wherein the primer sequence is as follows:
GhGT-3b_A04-F(SEQ ID No.3):
5’GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGAGGGACATCATCGC3’;
GhGT-3b_A04-R(SEQ ID No.4):
5’GGGGACCACTTTGTACAAGAAAGCTGGGTACATTTGATCTTCTCTTCT3’。
(2) Total RNA extraction from cotton leaves of upland cotton TM-1
The young leaves were ground to powder with liquid nitrogen and then total RNA of leaves was extracted using kit R FastPure Plant Total RNAIsolation Kit (Nuo Wei Zan, nanjing, china). RNA concentration and quality were measured by spectrophotometry at approximately 600ng/ul, OD260/OD280 = 2.0 and OD260/OD230 = 2.0, and the total RNA extracted was immediately used in the next experiment.
(3) RNA reverse transcription
The RNA was reverse transcribed into cDNA using a kit HISCRIPTIIIRT SUPERMIX FOR QPCR (Nuo-uzan, nanjing, china) from 1. Mu.g RNA to 0.2ml RNAase-free centrifuge tube, and the DNA concentration and quality were detected by a spectrophotometer at a concentration of about 1000 ng/. Mu.l, OD260/OD 280=1.8, OD260/OD 230=2.0, and the resulting cDNA was subjected to the next experiment.
(4) PCR amplification and purification
The PCR amplification was performed using high fidelity enzymes, specifically using a PCR reaction system of 1000 ng/. Mu.l cotton cDNA 1. Mu.l, 10. Mu.M primers F and R to 2. Mu.l each, 2X Phanta MasterMix: 25. Mu.l, ddH 2 O20. Mu.l, and a total system of 50. Mu.l. The PCR amplification conditions were specifically 95℃pre-denatured for 2 minutes, 95℃denatured for 15 seconds, 55℃annealed for 15 seconds, 72℃extended for 40 seconds, 35 cycles, 1% agarose gel electrophoresis separation and Gelred staining to detect the amplification results. The PCR purification and recovery were performed using Omega PCR purification and recovery kit to a concentration of 100 ng/. Mu.l, and ligation was performed.
(5) PCR fragment ligation expression vector
The expression vector construction was performed by using Gateway system, and the PCR product was reacted with the entry vector pDONR-Zeo in the BP reaction system of 1. Mu.l of pDONR-Zeo vector, 1. Mu.l of PCR product, 0.5. Mu.l of BP reaction enzyme (Invitrogen, california, USA) at 25℃for 2 hours to obtain pDONR-Zeo-GhGT-3b_A04 entry vector. The entry vector was sent to Shanghai Bioengineering Co.Ltd for sequencing, and the vector contained the nucleotide sequence shown as SEQ ID No.1, and the protein sequence contained a typical triple helix domain. Then, the entry vector was subjected to LR reaction with the objective vector pEarley Gate101 in a reaction system of 1. Mu.l of pDONR-Zeo-GhGT-3b_A04 vector, 1. Mu.l of PCR product, 0.5. Mu.l of LR-reactive enzyme (Invitrogen, california, U.S.A.), and 25℃for 1 hour to obtain the overexpression vector pEarley gate 101-GhGT-3b_A04. The pEarley gate 101-GhGT-3b_A04 vector was transformed into GV3101 Agrobacterium and positive colonies were identified by PCR.
(6)Real-time PCR
Extraction of RNA from upland cotton J11 and middle plant cotton No. 2 ZZ2 verticillium wilt infection 0h, 6h, 12h, 24h, 48h, 72h root and leaf and each tissue of upland cotton TM-1, reverse transcription into cDNA, real-time PCR using SYBR qPCR MasterMix, expression of GhGT-3b_A04 gene in root and leaf of J11 and ZZ2 was induced by verticillium (FIG. 1A-B), and relatively high expression in root, stem, petal and stamen of TM-1 (FIG. 1C).
Example 2
Obtaining transgenic Arabidopsis thaliana
(1) Transformation of Arabidopsis thaliana
Placing wild arabidopsis Col0 into a refrigerator at a temperature of 4 ℃ for vernalization treatment for 4 days, uniformly stirring nutrient soil and vermiculite according to a ratio of 3:1, adding water for wetting, then subpackaging into small basins, and directly sucking Col0 seeds into the small basins by using a gun head, wherein 9 seeds are planted in each basin. Arabidopsis thaliana was placed in a greenhouse at 24℃for cultivation in 16h of light, 8h of darkness. Culturing for about 45 days, and transforming when the buds are more. 200. Mu.l of pEarley gate 101-GhGT-3b_A04 positive Agrobacterium obtained in example 1 was cultured overnight in 2ml of LB+rif+kan medium at 28℃and 200rpm, 1ml of the bacterial liquid was grown in 100ml of the corresponding medium, grown for 12 hours at 28℃and 200rpm, and the bacterial liquid was collected at 4000rpm for 10 minutes, resuspended in a solution (1/2MS+5% sucrose+0.05% silwetl-77) and OD600 was adjusted to 1.5. And (3) placing the prepared dye-dip solution into an inverted culture dish, soaking the Arabidopsis flower batting in the dye-dip solution for 1min, horizontally placing the Arabidopsis plants after conversion, covering with a preservative film for moisturizing, placing the Arabidopsis plants in a greenhouse for cultivation after dark treatment for 24 hours, collecting seeds after the Arabidopsis is firm, and carrying out resistance screening on transgenic positive plants.
(2) Screening and identification of Positive plants
Uniformly broadcasting the obtained transgenic Arabidopsis T0 generation seeds in a nutrient soil matrix, and after the seeds germinate, starting to spray Basta herbicide once every 5 days, wherein the total spraying is 3 times. A total of 20 resistant plants are obtained, three resistant Arabidopsis genome DNA and RNA are extracted by using a CTAB method, target gene PCR identification and semi-quantitative PCR are carried out, the identified plants are positive plants, and the positive plants are expressed at a high RNA level and are respectively named GhGT-3b_A04-YFP-1-3 (A in figure 2). Since GhGT-3b_A04 and yellow fluorescent protein YFP are subjected to fusion expression, further western blot detection is performed on GhGT-3b_A04-YFP-1-3 by using an anti-GFP antibody, and as a result, it is found that all GhGT-3b_A04-YFP proteins of three strains are expressed (B in FIG. 2). Meanwhile, by taking the empty transgenic strain as a control, the phenotype of the arabidopsis thaliana 28-day plant is observed, and the overexpression strain is smaller than that of the plant of the control material (C in fig. 2), and the leaf cells are smaller when observed by a scanning electron microscope. By confocal laser observation of the Arabidopsis transgenic lines, no load was observed to be expressed in the cell membrane, nucleus and cytoplasm, whereas fluorescence of the GhGT-3b_A04-YFP transgenic lines was determined to be distributed only in the nucleus by DAPI staining (FIG. 3).
Example 3
Transgenic arabidopsis thaliana is more resistant to verticillium wilt
After the transgenic T3 generation homozygous strain is cultured in a greenhouse with 16h illumination, 8h darkness and 24 ℃ for 35 days, verticillium infection is carried out, and after 20 days, the phenotype of the transgenic strain is observed, and the leaf yellowing degree of three over-expressed transgenic strains GhGT-3b_A04-YFP-1-3 is lower than that of the wild type, which indicates that the transgenic strain is more resistant to verticillium than the wild type (figure 4). DAB staining of rosette leaves 24 and 48 hours from verticillium infection was observed to stain more deeply than wild-type leaves (FIG. 5), indicating that the transgenic lines could produce more reactive oxygen species to combat verticillium after verticillium infection.
Example 4
Root hair and epidermal hair changes in transgenic Arabidopsis thaliana
To analyze the root hair changes of transgenic arabidopsis, transgenic homozygous lines and control arabidopsis seeds were sterilized, vernalized at 4 ℃ and sown to 1% sucrose 1/2MS solid medium, and after 10 days of light culture, the root length and root hair number were observed by photographing. It can be seen that the GhGT-3b_A04-YFP-1-3 transgenic lines were higher in root hair length and root hair density than the wild type (FIG. 6).
After the transgenic T3 generation homozygous strain was cultured in a greenhouse at 24 ℃ for 28 days in 16h light and 8h darkness, the leaf epidermis hair phenotype of the transgenic strain was observed, and the epidermis hair density of the transgenic strain was observed to be higher than that of the wild type strain (FIG. 7).
Example 5
SA signal and synthesis path of transgenic arabidopsis thaliana and induced expression of disease-resistant related gene
Transgenic T3 generation homozygous line, after culturing for 28 days in a 16H light, 8H dark and 24 ℃ greenhouse, rosette RNA is extracted, reverse transcription is performed to cDNA, real-time PCR is performed, the expression of salicylic acid signal and synthesis pathway genes CBP60G, ICS and PBS3 are detected, all three genes are induced to be expressed in the transgenic line (A, B and G in figure 8), and simultaneously, the expression of disease resistance related genes PR2, CHI, CYP71A13, FRK1 and PNP-A, EARLI1 are detected, all six genes are induced to be expressed in the transgenic line (C-F, H-I in figure 8), which shows that GhGT-3b_A04 enhances verticillium resistance by activating salicylic acid signal and synthesis pathway and expression of disease resistance genes.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. The application of the over-expressed cotton GhGT-3b_A04 gene in improving the verticillium wilt resistance of arabidopsis thaliana is provided, and the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown as SEQ ID No. 1.
2. The application of the over-expressed cotton GhGT-3b_A04 gene in improving the root length of arabidopsis thaliana is provided, and the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown as SEQ ID No. 1.
3. The application of the over-expressed cotton GhGT-3b_A04 gene in improving the root hair density of arabidopsis thaliana is provided, and the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown as SEQ ID No. 1.
4. The application of the over-expressed cotton GhGT-3b_A04 gene in improving the epidermal hair density of arabidopsis thaliana is provided, and the nucleotide sequence of the cotton GhGT-3b_A04 gene is shown as SEQ ID No. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211311985.0A CN115786365B (en) | 2022-10-25 | 2022-10-25 | Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211311985.0A CN115786365B (en) | 2022-10-25 | 2022-10-25 | Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115786365A CN115786365A (en) | 2023-03-14 |
| CN115786365B true CN115786365B (en) | 2024-11-29 |
Family
ID=85433684
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211311985.0A Active CN115786365B (en) | 2022-10-25 | 2022-10-25 | Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115786365B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117327718B (en) * | 2023-12-01 | 2024-02-13 | 三亚中国农业科学院国家南繁研究院 | GhCRLK 1L104 gene and application, protein, over-expression vector and method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2402417T3 (en) * | 2003-08-11 | 2013-05-03 | Kweek-En Researchbedrijf Agrico B.V. | Fungus resistant plants and their uses |
| BR112012022218A2 (en) * | 2010-03-04 | 2018-07-10 | Mendel Biotechnology Inc | transcription regulators to improve plant performance |
-
2022
- 2022-10-25 CN CN202211311985.0A patent/CN115786365B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN115786365A (en) | 2023-03-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109456982B (en) | Application of rice OsMYB6 gene and encoding protein thereof in drought resistance and salt resistance | |
| CN110643618A (en) | Jatropha MYB transcription factor JcMYB16 gene and its application in improving plant drought resistance | |
| CN107299103B (en) | Thick boisiana IpASR gene and its coding albumen and application | |
| CN113234729B (en) | Gene GauRev2 capable of obviously improving verticillium wilt resistance of cotton and application thereof | |
| CN111944030A (en) | Wheat stress resistance regulatory protein TaCOR58 and coding gene and application thereof | |
| CN109879947B (en) | Moso bamboo transcription factor PheDof 2 gene and its application | |
| CN112725351A (en) | Application of gene OsWRKY43 in resisting bacterial blight of rice | |
| CN115786365B (en) | Cotton GhGT-3b_A04 gene, protein, expression vector, transgenic plant acquisition method and application | |
| CN114835789B (en) | Wheat powdery mildew resistance related protein TaGLP-7A, and coding gene and application thereof | |
| CN112126655A (en) | Application of Asian cotton GaNCED3 gene in improving drought resistance of plants | |
| CN110093353B (en) | Cold-resistant related coding gene of ordinary wild rice in bud stage and application thereof | |
| CN108424920B (en) | Maize reverse tolerance related transcription factor ZmNAC33 gene and its application | |
| CN107937417B (en) | A disease-resistant and drought-tolerant protein gene GhSNAP33 from cotton and its application | |
| CN111423500B (en) | SiMYB56 protein and application of encoding gene thereof in regulation and control of plant drought resistance | |
| CN102477435A (en) | Method for improving drought resistance of plant by using Poncirus trifoliata transcription factor gene PtrABF | |
| CN109734784B (en) | Application of SlDALR1 gene in enhancing resistance to bacterial leaf spot of tomato | |
| CN108034662B (en) | Application of wheat stripe rust PSTG _06025 gene in stripe rust prevention and treatment and cultivation method of stripe rust resistant wheat | |
| CN115340995B (en) | Mint drought-enduring gene McWRKY-like and expression protein and application thereof | |
| CN106397558B (en) | Application of protein and coding gene thereof in regulating and controlling verticillium wilt resistance of plants | |
| CN103789325A (en) | Cotton cell wall extensin gene GbEXPATR and application | |
| CN114214333A (en) | Gene for regulating and controlling development of plant leaf epidermal hair and thickness of secondary wall and application thereof | |
| CN109355270B (en) | A rice kinase OSK1 and its application | |
| CN106399327A (en) | Western balsam poplar PtrZFP103 gene as well as coding protein and application thereof | |
| CN108866074B (en) | Application of herbicide-resistant gene PAR3(G311E) | |
| CN118406669B (en) | Thiol peroxidase coding gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |