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Cotton WRKY transcription factor GarWRKY22 for regulating salt tolerance of plants and application thereof

A technology of transcription factor and salt tolerance, applied in the field of biological genetic engineering, can solve the problems of few research reports and achieve the effect of reducing salt tolerance

Active Publication Date: 2015-02-04
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, members of the WRKY transcription factor family have been found in a variety of plants, and the functions and regulatory networks of this transcription factor in plant response to adversity stress have been analyzed in the study of some model plants. Many WRKY transcription factors have also been found in cotton , but studies on WRKY transcription factors related to salt tolerance in cotton are rarely reported

Method used

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  • Cotton WRKY transcription factor GarWRKY22 for regulating salt tolerance of plants and application thereof
  • Cotton WRKY transcription factor GarWRKY22 for regulating salt tolerance of plants and application thereof
  • Cotton WRKY transcription factor GarWRKY22 for regulating salt tolerance of plants and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, the acquisition of GarWRKY22 gene

[0023] 1.1 Extraction of RNA

[0024] extract RNA

[0025] (1) Take 0.5g of fresh cotton tissue, add 0.1g of cross-linked polyvinylpyrrolidone (PVPP), fully grind to powder in liquid nitrogen, quickly transfer the frozen powder into a 10ml centrifuge tube, add 5ml of CTAB extract With 500 μL of 0.1M Tris-HCl of pH 8.0, bathe in water at 65°C for 20 minutes, and mix well by inverting halfway;

[0026] (2) Add an equal volume of chloroform to mix well, and let stand in an ice bath for 10 minutes;

[0027] (3) Centrifuge at 10,000 rpm for 20 minutes at 4°C. Divide into four 1.5ml centrifuge tubes;

[0028] (4) Aspirate the supernatant, add 1 / 3 volume of 8M LiCl and mix, -70°C for 30min or -20°C overnight;

[0029] (5) Centrifuge at 10,000 rpm for 20 minutes at 4°C. Discard the supernatant, wash twice with 70% ethanol, dry the precipitate and dissolve it in 30 μL DEPC water;

[0030] (6) Add 10U DNase without RNase ac...

Embodiment 2

[0062] Embodiment 2, the construction of plant expression vector

[0063] 2.1 Construction of pCAMBIA2301-CaMV35S-GarWRKY22 plant expression vector

[0064] Plant expression vector pCAMBIA2301-CaMV35S plasmid (Feng Juan et al., 2013; Cloning and functional analysis of the protein kinase gene GarCIPK8 of the wild species of Gossypium upland). BamHI and KnpⅠ were used to digest pCAMBIA2301-CaMV35S and the target gene fragment GarWRKY22 respectively, recover the large vector fragment and the target gene fragment, and transform E. coli Trans1-T1 competent cells (purchased from Beijing Quanshijin Biology Co., Ltd.) after ligation with T4 ligase. Technology Co., Ltd.), and the plant expression vector with the gene of interest was obtained after the recombinant was identified.

[0065] Enzyme digestion of pCAMBIA2301-CaMV35S plasmid and target gene fragment

[0066] The plasmid double enzyme digestion system is as follows:

[0067]

[0068]

[0069] Enzyme digestion at 30°C,...

Embodiment 3

[0086] Embodiment 3, preparation and transformation of Agrobacterium competent

[0087] 3.1 Preparation of Competent Agrobacterium EHA105

[0088] (1) Pick a single colony of EHA105, inoculate it in 5ml LB liquid medium, and culture overnight at 28°C with shaking at 200rpm until the OD600 value is 0.4;

[0089] (2) Inoculate in 400-500ml LB medium (in a 1L Erlenmeyer flask) at a ratio of 1:100, shake the bacteria until the OD600 is 0.6-0.8, and bathe in ice for 10 minutes;

[0090] (3) Collect the bacterial solution in a pre-cooled 50ml centrifuge tube, centrifuge at 5000rpm at 4°C for 5min;

[0091] (4) Discard the supernatant, fully suspend the precipitate with sterile water, centrifuge at 5000 rpm at 4°C for 5 min; repeat this process 3 times.

[0092] (5) Add 1 ml (depending on the number of bacteria) to the washed cells to resuspend the cells containing 10% sterile glycerol.

[0093] (6) Aliquot into 50 μL tubes, quick freeze in liquid nitrogen, and store at -80°C for ...

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Abstract

The invention discloses a transcription factor related to salt tolerance of cotton, namely gossypium wild species dry land cotton WRKY gene GarWRKY22 of which the sequence is disclosed as SEQ ID NO.2 in the sequence table. Electronic cloning and RT-PCR (reverse transcription-polymerase chain reaction) techniques are utilized to separate the protein GarWRKY22 related to salt tolerance of cotton. The functional verification by converting Arabidopsis thaliana proves that the transcription factor can regulate the salt tolerance. When the gene is overexpressed, the salt tolerance of the plants is obviously lowered.

Description

technical field [0001] The invention belongs to the technical field of biological gene engineering, and relates to a cotton transcription factor GarWRKY22 which negatively regulates plant salt tolerance. Background technique [0002] Soil salinization is a worldwide problem affecting agricultural production, and it is an important factor affecting agricultural output and leading to agricultural failure. The area of ​​saline soil in the world is about 1 billion hectares 2 , my country has a vast territory and a long coastline. There are a large number of coastal saline-alkali lands and inland saline-alkali lands. The total area of ​​saline-alkali soil in my country is about 100 million hm 2 , there are 6.7 million hectares of saline-alkali cultivated land and 20 million hectares of saline-alkali wasteland to be developed, and this figure is still rising. In addition, the production potential of secondary salinized land in northern irrigated areas needs to be further tapped. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00
CPCC07K14/415C12N15/8205C12N15/8273
Inventor 沈新莲范昕琦徐鹏郭琪张香桂
Owner JIANGSU ACAD OF AGRI SCI
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