A heavy metal-binding protein gene derived from high-temperature resistant bacteria and its anti-heavy metal application

A protein-binding and anti-heavy metal technology, applied in the field of genetic breeding, can solve the problems of destroying soil structure, secondary pollution, and high cost

Inactive Publication Date: 2017-03-29
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • 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, leading to / secondary pollution and other reasons, it is difficult to large area application

Method used

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  • A heavy metal-binding protein gene derived from high-temperature resistant bacteria and its anti-heavy metal application
  • A heavy metal-binding protein gene derived from high-temperature resistant bacteria and its anti-heavy metal application
  • A heavy metal-binding protein gene derived from high-temperature resistant bacteria and its anti-heavy metal application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Artificial Synthesis of Heavy Metal Binding Protein TtHMBP Gene of Thermostable Bacteria

[0036] According to the gene sequence of the thermostable bacteria TtHMBP registered in Genbank, the gene synthesis method [NucleicAcids 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) 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 (Tobias1991) to improve the stability of the translated protein. (5) The design increases the free energy at the 5' end of the g...

Embodiment 2

[0040] Construction of TtHMBP Agrobacterium Binary Vector

[0041] The positive clone of the artificially synthesized TtHMBP gene above was amplified by PCR with primers, then added BamHI 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 with the corresponding enzyme-cut vectors and digested with enzymes. The correct binary vector was obtained after 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 MicroPulserTM Electroporation Apparatus Operating Instructions and Application Guide (BIO-RAD Company) ((Rainerie et al., Bio. Tech., 1990, 8: 33-38).

[0045] 2) Take 50 μL of LGV3101 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

According to the invention, heavy metal binding protein originated from a high temperature-resistant bacterium is transformed into a plant, and such a transgenic plant is allowed to have an effect on improving resistance of the plant to heavy metals. The heavy metal binding protein gene is synthesized according to preferred codons of the plant and containing 213 bases and 68 coded amino acids. The nucleotide sequence of the gene is as shown in SEQ ID No. 1, and the amino acid sequence coded by the gene is as shown in SEQ ID No. 2. The heavy metal binding protein gene originated from the high temperature-resistant bacterium is synthesized by using an artificial method, and the transgenic plant has 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 high-temperature-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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N15/82C07K14/195A01H5/00
Inventor 薛永姚泉洪彭日荷朱波韩红娟韩静王波王红娟赵伟
Owner SHANGHAI ACAD OF AGRI SCI
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