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Acrylamide hydrolase gene derived from Burkholderia and application thereof

A technology of acrylamide and hydrolase, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2014-04-30
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no literature reports on acrylamide phytoremediation, so it is necessary to discover new acrylamide degradation methods

Method used

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  • Acrylamide hydrolase gene derived from Burkholderia and application thereof
  • Acrylamide hydrolase gene derived from Burkholderia and application thereof
  • Acrylamide hydrolase gene derived from Burkholderia and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Artificial synthesis of acrylamide hydrolase gene ACMDI

[0030] According to the PTDS (PCR-based two-step DNA synthesis, PTDS) method, the acrylamidase gene (ACMDI) from Burkholderia was cloned and chemically synthesized, on the basis of keeping the amino acid sequence of the ACMDI gene unchanged , design primers to synthesize the acrylamide hydrolase gene (ACMDI) of the present invention. The designed primers are as follows:

[0031] 1. Acmdi-1: Tm=54, 60mer

[0032] TCA, GTC, TTC, GTG, CGG, GAT, TCC, TTC, GAT, CGG, ACA, CTC, CGG, CGT, GCC, AGG, GGT, TGG, ACG, CGT

[0033] 2. Acmdi-2: Tm=54, 60mer

[0034] TGT, GGA, TCC, AGA, CGC, CAC, GCG, TCG, CCG, CGT, CGA, AGC, GCT, CAC, GCG, TCC, AAC, CCC, TGG, CAC

[0035] 3. Acmdi-3: Tm=54, 60mer

[0036] GCG, TGG, CGT, CTG, GAT, CCA, CAA, TCC, AGT, CGC, GAT, AGA, AGC, CGA, ATG, GAC, AGT, CCG, CGA, CGC

[0037] 4. Acmdi-4: Tm=54, 60mer

[0038] GGT, TAT, ACC, GGC, ACC, ATT, CAT, TCC, GGC, GAT, GGC, GTG, AAT, GG...

Embodiment 2

[0085] The construction of embodiment 2 ACMDI Agrobacterium binary vector

[0086] The ORF of the above-mentioned artificially synthesized ACMDI gene was amplified by PCR, and the head and tail were respectively added with Bam HI and Sac I cut points, and after Bam HI+Sac I double enzyme digestion, the recovered DNA fragment was connected to the corresponding enzyme-cut vector, identified by enzyme digestion and The correct binary vector pYM6032 was obtained after sequencing.

Embodiment 3

[0087] Example 3 Transformation of Agrobacterium by electric shock method

[0088] 1) Preparation of Agrobacterium GV3101 competent, the method refers to MicroPulser TM Electroporation Apparatus Operating Instructions and Application Guide (BIO-RAD Corporation) (Raineri et al., 1990).

[0089] 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).

[0090] 3) Pick several clones from the bacteria grown on the LB plate in step 2, extract the Agrobacterium plasmid by alkaline method, identify by enzyme digestion, and detect by PCR.

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Abstract

The invention relates to an acrylamide hydrolase gene derived from Burkholderia and application thereof. The acrylamide hydrolase gene is synthesized using artificial method and has a nucleotide sequence shown in SEQ ID NO.1, and the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO.2. The results of Agrobacterium-mediated transformation of the acrylamide hydrolase gene into Arabidopsis thaliana using floral dip method shows that the transgenic Arabidopsis thaliana successfully has the function of decomposing acrylamide with the help of the acrylamide hydrolase gene, which makes remediation of acrylamide pollution with other plants possible.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically relates to an acrylamide hydrolase gene derived from Burkholderia bacterium and its application. Background technique [0002] Acrylamide (CH 2 =CH-CONH 2 , Acrylamide) is a white crystalline substance with a molecular weight of 70.08, which has been widely used in the production of chemical products polyacrylamide precursors since 1950. Polyacrylamide is mainly used for water purification, pulp processing and inner coating of pipelines. Due to its potential neurotoxicity, genotoxicity and carcinogenicity, acrylamide poses a great threat to human health. For this reason, how to degrade the residual acrylamide in the environment is an urgent problem to be solved. [0003] Phytoremediation is the use of green plants to transfer, accommodate or transform pollutants to make them harmless to the environment. The object of phytoremediation is soil and water polluted by hea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55
Inventor 高建杰姚泉洪彭日荷熊爱生付晓燕田永生赵伟金晓芬韩红娟陈晨
Owner SHANGHAI ACAD OF AGRI SCI