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A kind of cotton ATP hydrolase atpase 2 and its coding gene and application

A technology of hydrolase and gene, applied in the field of cotton ATP hydrolase ATPase‑2 and its coding gene and application, capable of solving problems such as complex mechanism and important issues of plant salt resistance to be explored

Inactive Publication Date: 2018-03-16
BIOCENTURY TRANSGENE CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although researchers have carried out a lot of research from different aspects, many important issues in plant salt resistance remain to be explored due to the complexity of its mechanism

Method used

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  • A kind of cotton ATP hydrolase atpase 2 and its coding gene and application
  • A kind of cotton ATP hydrolase atpase 2 and its coding gene and application
  • A kind of cotton ATP hydrolase atpase 2 and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Cotton SSH library construction under salt stress:

[0019] The specific method is:

[0020] According to Clontech's PCR-select TM The method shown in the cDNA Subtraction Kit kit manual was used to construct the SSH library (suppression subtraction library) by the suppression subtractive hybridization method. During the experiment, the mRNA extracted from the salt-treated cotton root tissue was used as the sample (Tester), and the mRNA extracted from the untreated cotton root tissue was used as the control (Driver). Specific steps are as follows:

[0021] (1) Test materials:

[0022] African cotton (National Cotton Medium-Term Bank, acquired by China Cotton Research Institute, unified number: ZM-06838) was sown on sterilized vermiculite, cultivated at 25°C, with a light-dark cycle of 16h / 8h, and watered 1 / 2 2MS medium (9.39mM KNO 3 , 0.625mM KH2PO 4 , 10.3mM NH 4 NO 3 , 0.75mM MgSO 4 , 1.5mM CaCl 2 , 50 μM KI, 100 μM H 3 BO 3 , 100 μM nSO 4 , 3...

Embodiment 2

[0033] Example 2 Cloning of cotton ATP hydrolase gene GhATPase-2

[0034] After removing redundant DNA from the clone of the identified cotton SSH library from colony Gh-S2-176, the sequence is SEQ ID No: 3, and sequence analysis shows that the protein encoded by this sequence belongs to ATP hydrolase. In this paper, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 is named GhATPase-2, and the corresponding protein is named ATPase-2.

[0035] SEQ ID No: 3:

[0036]

[0037]

[0038] Cloning of the full-length coding gene of GhATPase-2

[0039] According to the obtained sequence of SEQ ID No: 3, the following two specific primers were designed as the 5' end specific primers of 3' RACE.

[0040] GhATPase-2 GSP1: SEQ ID No: 4:

[0041] GTAGAAGCTT GCATAAATGC TG

[0042] GhATPase-2 GSP2: SEQ ID No: 5:

[0043] ACAGCAAAGG CTATAGCAAC TGA

[0044] The experimental steps were operated according to the instructions of the kit (the 3'RACE System for R...

Embodiment 3

[0079] The construction of the plant expression vector of embodiment 3 GhATPase-2 gene

[0080] The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as the plant expression vector, and the 35S promoter of the NPTII gene containing double enhancers was replaced with the Pnos promoter to reduce the expression of NPTII protein in plants . The 35S promoter and the Tnos terminator were selected as the promoter and terminator of the GhATPase-2 gene, respectively, and the construction flow chart is shown in Figure 1.

[0081] Primers SEQ ID NO: 12 and SEQ ID NO: 13 were used to amplify Pnos with the plant expression vector pBI121 plasmid (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 μl PCR reaction system: 10 μl 5×PS Buffer, 3 μl 2.5 mM dNTP, 1.0 μl pBI121 plasmid, 1.0 μl PrimeSTAR HS DNA polymerase, 10 μM primers SEQ ID NO: 12 ...

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Abstract

A plant protein and its encoding gene and application are provided, especially an ATP hydrolase derived from cotton and its encoding gene, as well as its application in cultivating transgenic plants with improved salt tolerance.

Description

technical field [0001] The invention relates to plant protein and its encoding gene and application, in particular to an ATP hydrolase ATPase-2 derived from cotton and its encoding gene, as well as its application in cultivating transgenic plants with improved salt tolerance. Background technique [0002] Salt stress is one of the most important abiotic stress hazards to agricultural production in the world. Saline soils are usually dominated by sodium, calcium or magnesium salts, which have become the main factors that affect plant growth and lead to reduced yields of food and economic crops. The area of ​​saline-alkali soil in the world is about 400 million hectares, accounting for 1 / 3 of irrigated farmland. Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 40 million hectares. With the increase of population and the reduction of cultivated land in our country, the development and utilization of saline-alkali land resourc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/29C12N15/08C12N5/04C07K14/415A01N65/08A01H1/00A01H4/00A01P21/00
CPCC12N9/16A01N65/00C12N15/8273
Inventor 何云蔚崔洪志王建胜田大翠梁丽
Owner BIOCENTURY TRANSGENE CHINA
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