Heavy metal binding protein gene derived from radio-resistant bacterium and application of gene in heavy metal resistance

A technology that binds proteins and anti-radiation bacteria, applied in the field of genetics and breeding, can solve the problems of low efficiency, damage to soil structure and high cost

Inactive Publication Date: 2015-02-11
南京柏欧美地生物科技有限公司
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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 me...

Method used

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  • Heavy metal binding protein gene derived from radio-resistant bacterium and application of gene in heavy metal resistance
  • Heavy metal binding protein gene derived from radio-resistant bacterium and application of gene in heavy metal resistance
  • Heavy metal binding protein gene derived from radio-resistant bacterium and application of gene in heavy metal resistance

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Experimental program
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Effect test

Embodiment 1

[0035] Synthesis of Heavy Metal Binding Protein DrHMBP Gene of Radiation Resistant Bacteria

[0036] According to the gene sequence of the radioresistant bacteria DrHMBP registered in Genbank, the gene synthesis method [Nucleic Acids Research, 2004, 32, e98] was used to design the coding region of the synthetic gene according to the following principles without changing the amino acid sequence encoded by the gene : (1) Optimizing 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 (Tobias1991) to improve the stability of the translated protein. (5) The design increases the free energy at the 5' end of the...

Embodiment 2

[0040] Construction of DrHMBP Agrobacterium Binary Vector

[0041] The positive clone of the artificially synthesized DrHMBP gene above was 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, and 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 ).

Embodiment 3

[0043] Transformation of Agrobacterium by electroporation

[0044]1) Preparation of 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).

[0045] 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, 25 μf). Add 1 mL of LB medium containing 1% mannitol to recover the culture for 2 hours (28° C., 250 rpm). Take 10 μL and 100 μL respectively and apply on LB plates (rifampicin 50 μg / mL, gentamicin 50 μg / mL, chloramphenicol 100 μg / mL).

[0046] 3) Pick several clones, extract the Agrobacterium plasmid by alkaline method, identify by enzyme digestion, and detect by PCR.

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Abstract

The invention discloses a heavy metal binding protein gene derived from radio-resistant bacterium and application of the gene in heavy metal resistance. According to the invention, a heavy metal binding protein derived from the radio-resistant bacterium is transformed to a plant, so that the transgenic plant has an effect of enhancing heavy metal resistance of the plant. The heavy metal binding protein gene is synthesized in accordance with plant preferred codons contains 219 basic groups and codes 71 amino acids; the nucleotide sequence of the gene is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2. The gene derived from the radio-resistant bacterium is synthesized by re-simplification in conjunction with artificial method; and the transgenic plant has relatively high heavy metal resistance.

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 radiation-resistant bacteria. The gene is transformed into plants by using Agrobacterium, so that the metal resistance ability of plants is improved. 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...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12N15/84A01H5/00C12R1/01
Inventor 周惠苗辉蒋海燕张杨薛永蒋小辉伍光彩
Owner 南京柏欧美地生物科技有限公司
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