Methylobacterium extorquens heavy metal binding protein MaHMBP, MaHMBP encoding gene, plant binary vector and application

A technology of Methylobacterium extorquens and binding proteins, which is applied in the field of genetic breeding and can solve the problems of restricting the application of phytoremediation and the limited ability of phytoremediation

Inactive Publication Date: 2017-07-21
SHANGHAI ACAD OF AGRI SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the ability of phytoremediation is currently limited. For example, plants have limited resistance to the types and concentrations of heavy metals, which greatly restricts the application of phytoremediation.

Method used

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  • Methylobacterium extorquens heavy metal binding protein MaHMBP, MaHMBP encoding gene, plant binary vector and application
  • Methylobacterium extorquens heavy metal binding protein MaHMBP, MaHMBP encoding gene, plant binary vector and application
  • Methylobacterium extorquens heavy metal binding protein MaHMBP, MaHMBP encoding gene, plant binary vector and application

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preparation example Construction

[0021] In the present invention, the synthesis method of the Methylobacterium extorquens heavy metal binding protein coding gene MaHMBP is preferably artificially synthesized. The manual method includes the following steps:

[0022] According to the PTDS (PCR-based two-step DNA synthesis, PTDS) method, the heavy metal binding protein coding gene derived from M. extorquens was chemically synthesized, and the coding region of the gene was synthesized on the basis of keeping the amino acid sequence of the MaHMBP gene unchanged. According to the obtained nucleotide sequence of gene coding region, primers were designed to amplify MaHMBP gene.

[0023] In the present invention, the design method of the coding region of the synthetic gene comprises the following parts:

[0024] (1) Optimizing gene codons to improve gene translation efficiency;

[0025] (2) Eliminate the recognition sites of commonly used restriction endonucleases inside the gene to facilitate the construction of ex...

Embodiment 1

[0052] Artificial Synthesis of Heavy Metal Binding Protein MaHMBP Gene of Methylobacterium extorquens

[0053] According to the Methylobacterium extorquens MaHMBP gene sequence registered in Genbank, the gene synthesis method [NucleicAcids Research, 2004, 32, e98] is used to synthesize the gene coding region according to the following principles without changing the amino acid sequence of the gene code Design: (1) Optimize gene codons to improve gene translation efficiency. (2) Eliminate the recognition sites of commonly used restriction endonucleases inside the gene to facilitate the construction of expression cassettes. (3) Eliminate inverted repeat sequences, stem-loop structures and transcription termination signals, balance GC / AT within genes, and improve RNA stability. (4) Make the gene-encoded protein conform to the N-terminal principle (Tobias 1991) to improve the stability of the translated protein. (5) The design increases the free energy at the 5' end of the gene ...

Embodiment 2

[0057] Construction of MaHMBP Agrobacterium Binary Vector

[0058] The positive clones of the artificially synthesized MaHMBP gene above were amplified by PCR with primers, then added Bam HI and Sac I cutting points at the head and tail, respectively, and double-digested with Bam HI+Sac I, the recovered DNA fragments were connected to the corresponding enzyme-cut vectors, The correct binary vector was obtained after cleavage identification and sequencing (see figure 1 ).

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Abstract

The invention provides the heavy metal binding protein MaHMBP of Methylobacillus extorquens and its encoding gene, plant binary vector and application, belonging to the field of gene breeding. The heavy metal binding protein MaHMBP of Methylobacter extorquens provided by the present invention and its coding gene are transferred into Arabidopsis thaliana to obtain transgenic Arabidopsis, and the heavy metal resistance of the Arabidopsis plants transfected with the MaHMBP gene and wild-type Arabidopsis plants are compared Studies have shown that the MaHMBP gene significantly improves the heavy metal resistance of Arabidopsis.

Description

technical field [0001] The invention belongs to the field of genetic breeding, and specifically relates to the heavy metal binding protein MaHMBP of Methylobacter extorquens and its encoding gene, a plant binary carrier and application. Background technique [0002] With the rapid development of modern industry and agriculture and the rapid expansion of cities, the heavy metal pollution in the natural environment is becoming more and more serious. Every year due to heavy metal pollution, the production of grain is reduced by more than 10 million tons, and the grain polluted by heavy metals reaches 12 million tons every year, and the annual economic loss is more than 20 billion. Traditional soil heavy metal pollution remediation technologies include landfill method, dilution method, leaching method, physical separation method and chemical method, etc. Due to high cost, low efficiency, and damage to the soil structure, resulting in secondary pollution, etc., it is difficult to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/84A01H5/00
CPCC07K14/195C12N15/8271
Inventor 薛永秦秦宋科乔红霞杨建军蔡进泉郑宪清李双喜吕卫光
Owner SHANGHAI ACAD OF AGRI SCI
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