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Heavy metal binding protein gene from Methylobacterium extorquens, and its anti-heavy metal application

A technology of Methylobacter extortion and protein binding, applied in the field of genetics and breeding, can solve the problems of destroying soil structure, high cost, difficult to apply in a large area, etc.

Inactive Publication Date: 2014-10-15
NANJING BIO GOOD SCIANDTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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 Area application

Method used

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  • Heavy metal binding protein gene from Methylobacterium extorquens, and its anti-heavy metal application
  • Heavy metal binding protein gene from Methylobacterium extorquens, and its anti-heavy metal application
  • Heavy metal binding protein gene from Methylobacterium extorquens, and its anti-heavy metal application

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Experimental program
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Effect test

Embodiment 1

[0036] heavy metal binding protein of Methylobacter extorquens wxya artificial synthesis of genes

[0037] Methylobacter extorquens registered according to Genbank M wxya Gene sequence is used for gene synthesis [Nucleic Acids Research, 2004, 32, e98]. Under the premise of not changing the amino acid sequence encoded by the gene, the coding region of the synthetic gene is designed according to the following principles: (1) Optimize the gene codon , 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) Designed to increase the free energy at the 5'...

Embodiment 2

[0041] wxya Construction of Agrobacterium Binary Vector

[0042] artificially synthesized wxyaThe primers for the positive clone of the gene were amplified by PCR, and the head and the tail were respectively added with Bam HI and Sac I cutting points, and after Bam HI+Sac I double enzyme digestion, the recovered DNA fragment was connected with the corresponding enzyme-cut carrier, and the correct DNA fragment was obtained after enzyme digestion identification and sequencing. binary vector (see figure 1 ).

Embodiment 3

[0044] Transformation of Agrobacterium by electroporation

[0045] 1) Prepare Agrobacterium GV3101 competent, the method refers to MicroPulser TM Electroporation Apparatus Operating Instructions and Application Guide (BIO-RAD Company) ((Raineri et al., Bio. Tech., 1990, 8: 33-38).

[0046] 2) Take 50 μL of GV3101 competent cells, add 1 μL of DNA, and transfer to a 0.2 cm electric shock cup for transformation (400Ω, 2.5KV, 25μf). Add 1 mL of LB medium containing 1% mannitol to recover the culture for 2 hours (28°C, 250rpm). Take 10 μL and 100 μL respectively and smear LB plates (rifampicin 50 μg / mL, gentamicin 50 μg / mL, chloramphenicol 100 μg / mL).

[0047] 3) Pick several clones, extract the Agrobacterium plasmid by alkaline method, identify by enzyme digestion, and detect by PCR.

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Abstract

A heavy metal binding protein gene from Methylobacterium extorquens is transferred to a plant to make the obtained transgenic plant have an increased anti-heavy metal effect. The the heavy metal binding protein gene synthesized according to plant biased codons contains 234 bases and codes 73 amino acids; and the nucleotide sequence of the gene is represented by SEQ ID NO:1, and the sequence of the amino acids coded by the gene is represented by SEQ ID NO:2. The heavy metal binding protein gene from Methylobacterium extorquens is artificially synthesized, and the transgenic plant has a high anti-heavy metal property.

Description

technical field [0001] The invention belongs to the field of genetic breeding, and specifically relates to a sequence of a heavy metal binding protein gene derived from Methylobacterium extorquens, which is transformed into plants by using Agrobacterium to improve the metal resistance ability of plants. 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 s...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12N15/84A01H5/00C12R1/01
Inventor 薛永蒋小辉徐小赟侯晓光吴顺霞侯曙光周惠伍光彩张扬
Owner NANJING BIO GOOD SCIANDTECH
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