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Tonoplast hydrogen pyrophosphatase gene of strong xerophyte zygophyllumxanthoxylum and plant expression vector as well as genetic plant transformation method thereof

A technology of xerophyte and tonoplast membrane, which is applied in the field of strong xerophyte overlord tonoplast hydrogen pyrophosphatase gene and plant expression vector and its plant genetic transformation, which can solve the problems of weak stress resistance and other problems

Inactive Publication Date: 2012-01-11
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] Regrettably, however, currently cloned tonoplast H + -PPase genes mostly come from model plants or crops with weak stress resistance, such as Arabidopsis, wheat, rice and tobacco (Bao Aike, Zhang Jinlin, Guo Zhenggang, et al. 2006. Plant Physiology Communications. 42(4): 777- 783); while H + -PPase has not been reported in desert xerophytes

Method used

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  • Tonoplast hydrogen pyrophosphatase gene of strong xerophyte zygophyllumxanthoxylum and plant expression vector as well as genetic plant transformation method thereof
  • Tonoplast hydrogen pyrophosphatase gene of strong xerophyte zygophyllumxanthoxylum and plant expression vector as well as genetic plant transformation method thereof
  • Tonoplast hydrogen pyrophosphatase gene of strong xerophyte zygophyllumxanthoxylum and plant expression vector as well as genetic plant transformation method thereof

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Effect test

Embodiment 1

[0034] Example 1: Bawang H + -The cloning method of PPase gene:

[0035] According to other plants H + -A pair of degenerate primers were designed for the conservative sequence of PPase gene, using the total RNA of Bawang leaf as the template, and using RT-PCR to amplify H + -PPase gene fragment and cloned into pUCm-T vector. The positive clones were identified by PCR and sequenced, and the sequence analysis results showed that: 3 homologous sequence fragments (898bp) were cloned, encoding 299 amino acids; the obtained sequence was the same as the higher plant H registered in GenBank + -The homology of the nucleotide sequence of the PPase gene is more than 69%, and the homology of the amino acid sequence is more than 78%. In addition, all three fragments contain vacuolar membrane H + -PPase unique binding site and active site sequence of PPi. There are two types of H in higher plants + -PPase: Type I depends on K + Activated and moderately Ca 2+ Activated, mainly distributed on t...

Embodiment 2

[0070] Example 2: ZxVP recombinant nucleic acid molecule vector

[0071] According to different production or experimental purposes, such as obtaining recombinant proteins or obtaining transgenic plants, different vectors and corresponding host cells and corresponding transformation methods can be selected for different practical applications. It should be pointed out that the nucleic acid molecule of the present invention is not limited to a specific expression vector, nor is it limited to a specific transformation host, nor is it limited to a specific transformation method.

Embodiment 3

[0073] Example 3: Bawang H + -Acquisition of PPase recombinant protein

[0074] Acquire Overlord H + -PPase protein or a part of the protein, the method described in "recombinant egg and avian adenovirus-based gene cloning and expression vector" (Chinese Patent No. 00815895.9) can be used to combine the nucleic acid molecule or nucleic acid of the present invention The molecular fragments were recombined into a chicken adenovirus expression vector AdCEV, and then the recombinant nucleic acid molecule was transfected into chicken eggs by microinjection, and then the transfected chicken eggs were placed in a humidified incubator at 37°C After 72 hours of culture, the allantoic fluid was collected and reconstituted H + -PPase protein or a part of the protein is separated and purified. The purification method can adopt common purification methods, such as ion exchange, affinity chromatography, immunoprecipitation, etc. or a combination of these methods. Of course, vectors suitable f...

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Abstract

The invention relates to tonoplast hydrogen pyrophosphatase gene ZxVP1-1 and two core segments ZxVP1-2 and ZxVP1-3 cloned from succulent xerophyte zygophyllumxanthoxylum peculiar to deserts in Central Asia, and a plant expression vector Pcambia1302-ZxVP1-1 of the gene as well as an engineering agrobacterium GV3101 containing the expression vector. The invention belongs to the technical field of molecular biology and biology, and is mainly used for genetic improvement of crops, can improve characteristics of salinity tolerance, drought control, cold resistance, leanness resistance, biomass andthe like of the crops, and has the important social benefit, economic benefit and biological benefit.

Description

Technical field: [0001] The present invention belongs to the field of molecular biology and biotechnology, and relates to the tonoplast hydropyrophosphatase gene ZxVP1-1 cloned from the ancient strong xerophyte (Zygophyllum xanthoxylum) unique to the desert of Central Asia, and the plant expression vector pCAMBIA1302 of the gene -ZxVP1-1 and engineered Agrobacterium GV3101 containing the expression vector and its plant genetic transformation method. Background technique: [0002] Drought and soil salinization are the two most important unfavorable factors restricting global agricultural and animal husbandry production. At present, the total area of ​​arid areas in the world is about 6.1 billion hectares, accounting for about 40% of the world's land area (Global Times 2009). Moreover, due to greenhouse gas emissions, the temperature rises and the evaporation of water intensifies, causing some semi-humid areas to become arid areas. In addition, due to the intense evaporation in a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N9/14C07K16/40C12N15/63C12N1/21C12N5/10A01H5/00C12N15/84
Inventor 王锁民席杰军伍国强王燕雯
Owner LANZHOU UNIVERSITY
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