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Na+/H+ antiporter protein Nha-K2 and encoding gene and application thereof

A technology of antiporter and coding gene, which is applied in the field of Na+/H+ antiporter Nha-K2 and its coding gene and application, can solve the problems of large investment, low benefit, aggravated soil secondary salinization, etc., and achieve improvement Adaptability, the effect of improving the ability to tolerate salt environments

Inactive Publication Date: 2012-06-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, traditional measures require large investment and low benefits, and the addition of a large number of chemical substances will aggravate the secondary salinization of the soil, which forces people to consider other measures to improve the use of saline soil

Method used

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  • Na+/H+ antiporter protein Nha-K2 and encoding gene and application thereof
  • Na+/H+ antiporter protein Nha-K2 and encoding gene and application thereof
  • Na+/H+ antiporter protein Nha-K2 and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of Keke Salt Lake Metagenome Library, Cloning and Sequence Analysis of nha-K2 Gene

[0041] (1) Construction of Keke Salt Lake Metagenome Library

[0042] Keke Salt Lake is located in the northeast of the Qaidam Basin, in a small basin under the Yak Mountain in Ulan County, Qinghai Province. The lake is 3010 meters above sea level, about 28 kilometers long from east to west, narrow from north to south, and covers an area of ​​119 square kilometers. The water of the lake is sodium-salt type, gradually drying up, and now there are only small areas of water at the east and west ends. The source of intercrystalline brine is the spring water that escapes from the clay layer after passing through the gravel layer of the sloped plain in front of the mountain. There are more than 665 spring holes. The reserves of salt mines are large, about 7.5 million tons per square kilometer, and the total reserves of sodium chloride are more than 900 million tons. I...

Embodiment 2

[0066] Example 2 Effect of Antiporter Nha-K2 on Improving Salt Tolerance of Escherichia coli

[0067] Through the sampling locations listed in Example 1, soil samples were taken, enriched and cultivated in 8% Gibbson medium, and after the total DNA was extracted, it was partially digested to construct a metagenomic library, and the Escherichia coli salt-sensitive mutant KNabc was used as the subject The body is transformed, and the nha-K2 gene can be obtained by screening in LBK medium containing 0.2M NaCl.

[0068] (1) Construction of recombinant plasmids

[0069] Plasmid pUC118 was digested with restriction endonuclease Sal I, and then it was connected with the fragment (about 2.7 kb) containing nha-K2 gene and its upstream and downstream sequences with the same restriction endonuclease to obtain recombinant plasmid pUCNha-K2 ( figure 1 ), it was identified that pUCNha-K2 inserted an exogenous fragment containing nha-K2 gene (such as SEQ ID NO.1 in the sequence listing) at ...

Embodiment 3

[0078] Example 3 Detection of monovalent cation reverse transport activity of Nha-K2 protein

[0079] (1) Preparation of reverse membrane

[0080] The so-called inverted membrane is a mixture of bacterial crude protein, because the inverted membrane of the positive clone E.coli KNabc / pUC Nha-k2 contains Nha-K2 protein, while the inverted membrane of the negative control bacteria E.coli KNabc / pUC118 The membrane does not contain Nha-K2 protein, and the function of Nha-K2 protein to transport salt ions can be determined by detecting the monovalent cation transport activity of the reverse membrane of these two bacteria.

[0081] The positive clone E.coli KNabc / pUC Nha-K2 obtained in Example 2 and the Escherichia coli E.coli KNabc / pUC118 (negative control) transferred to the empty vector pUC118 were inoculated in an Erlenmeyer flask filled with 20ml LBK liquid medium, Cultivate overnight in a shaker at 37°C and 180r / min. Then, they were transferred to 200 mL of LBK liquid medium...

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Abstract

The invention discloses Na+ / H+ antiporter protein Nha-K2 and encoding gene and application thereof. The encoding gene of the Na+ / H+ antiporter protein Nha-K2 has the 571st to 2058th of the SEQ (sequence) ID NO.1 nucleotide sequence in the sequence table. The Na+ / H+ antiporter protein Nha-K2 has higher antiporter protein activity, is capable of improving haloduric performance of host bacteria after being led into the host bacteria to express, can also be transferred into crops so that transgenosis crops with haloduric performance are obtained and adaptability of the crops can be improved, and accordingly has important application prospect in breeding of the crops.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Na + / H + Antiporter Nha-K2 and its coding gene and application. Background technique [0002] Due to global warming, increasing world population, increasing industrial pollution, development of irrigated agriculture, improper use of chemical fertilizers and other factors, soil salinization is becoming more and more serious, which not only makes soil desertification and ecological environment worse, but also has become a hindrance to crop growth. The main factor of growth and high yield and quality. At present, the main way to solve the problem is the traditional method of improving saline soil, such as fresh water washing, rational irrigation and the use of CaCO 3 Wait. However, the traditional measures require large investment and low benefits, and the addition of a large number of chemical substances will aggravate the secondary salinization of the soil, which forces people ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/63C12N15/70C12N1/21C12N15/84A01H5/00C12R1/19
Inventor 陈三凤高淼陶丽王磊
Owner CHINA AGRI UNIV
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