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A sodium-hydrogen antiporter protein nhx2 in the tonoplast membrane of Saltina japonicus and its coding gene and application

An antiporter and tonoplast technology, applied in the field of plant proteins and their encoding genes and applications, can solve problems such as complex mechanisms and important issues of plant salt resistance yet to be explored.

Inactive Publication Date: 2018-11-09
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 sodium-hydrogen antiporter protein nhx2 in the tonoplast membrane of Saltina japonicus and its coding gene and application
  • A sodium-hydrogen antiporter protein nhx2 in the tonoplast membrane of Saltina japonicus and its coding gene and application
  • A sodium-hydrogen antiporter protein nhx2 in the tonoplast membrane of Saltina japonicus and its coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1. Construction of the SSH library of Salt mustard under salt stress:

[0020] The specific method is:

[0021] According to PCR-select of Clontech TM The method shown in the instructions of the cDNA Subtraction Kit kit uses the suppression subtractive hybridization method to construct an SSH library (subtractive library). During the experiment, the mRNA extracted from the salt-treated salt mustard tissue was used as the sample (Tester), and the mRNA extracted from the untreated salt mustard tissue was used as the control (Driver). Specific steps are as follows:

[0022] (1) Test materials:

[0023] Small salt mustard (Thellungiella halophila, purchased from the Halophyte Breeding Center of Ulanbuhe Desert Green Botanical Garden, Bayannur City, Inner Mongolia, China) was sown on sterilized vermiculite, at 22℃, photoperiod 12 hours light / 12 hours dark ( Culture under the condition of light intensity 3000-4000Lx), pour 1 / 2MS medium (containing 9.39mM KNO 3 , 0.625mMK...

Embodiment 2

[0034] Example 2 Cloning of tonoplast sodium hydrogen antiporter gene ThNHX2

[0035] After removing the redundant DNA of the clone from the colony Th-S312 in the identified salt mustard SSH library, the sequence is SEQ ID No: 3. Sequence analysis shows that the protein encoded by the sequence belongs to the vacuolar membrane sodium hydrogen antiporter . In this paper, the full-length coding gene corresponding to the SEQ ID No: 3 sequence is named ThNHX2, and its corresponding protein is named NHX2.

[0036] SEQ ID No: 3:

[0037]

[0038] Cloning of the full-length encoding gene of ThNHX2

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

[0040] ThNHX2GSP1: SEQ ID No: 4:

[0041] GCGAGGGCAG CTAATGTGTT TG

[0042] ThNHX2GSP2: SEQ ID No: 5:

[0043] ACCTGCTCAT AGGAAAATAC CA

[0044] The experimental steps were performed according to the kit instructions (3'RACE System for Rapid Amplification ...

Embodiment 3

[0076] Example 3 Plant expression vector construction of ThNHX2 gene

[0077] 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 with double enhancers was replaced with the Pnos promoter to reduce the expression of NPTII protein in plants . The 35S promoter and Tnos terminator were selected as the promoter and terminator of the ThNHX2 gene, respectively. The construction flow chart is shown in Figure 1.

[0078] Using primers SEQ ID NO: 12 and SEQ ID NO: 13, the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) was used as a template to amplify Pnos, and PrimeSTAR HS DNA polymerase of TaKaRa was used. 50μl PCR reaction system: 10μl 5×PS Buffer, 3μl 2.5mM dNTP, 1.0μl pBIl21, 1.0μl PrimeSTAR, 10μM primers SEQ ID NO: 12 and SEQ ID NO: 13 each 2.0μl and 31μl double distilled water. PCR reaction cond...

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Abstract

Disclosed are a tonoplast sodium-hydrogen anti-protein NHX2 derived from Thellungiella halophila and coding gene thereof, and use thereof in the cultivation of transgenic plants having improved salt tolerance.

Description

Technical field [0001] The present invention relates to plant protein and its encoding gene and application, in particular to a vacuolar membrane sodium hydrogen antiporter NHX2 and its encoding gene derived from salt mustard, and its application in cultivating transgenic plants with improved salt tolerance . Background technique [0002] Salt stress is one of the most important abiotic stress hazards in agricultural production in the world. Saline soil is usually dominated by sodium, calcium or magnesium, which has become the main factor affecting plant growth and leading to reduced production of food and cash 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. As the population of our country increases and cultivated land decreases, the development and utilization of saline-alkali la...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N15/82A01H4/00A01H5/00A01H6/20
CPCC07K14/415
Inventor 崔洪志梁远金田大翠刘捷
Owner BIOCENTURY TRANSGENE CHINA
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