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Cloning and Characterization of a Novel Gene Involved in Glyphosate Degradation

Author: DaiChunZuo
Tutor: HaoDongYun
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: Metagenomic library Glyphosate -degrading enzyme Glycine oxidase Clone Heterologous expression and purification Enzymatic properties of
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 81
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Abstract

Glyphosate is a broad spectrum off naturally, within the suction excellent conductivity herbicides. Its mechanism of action is competitive inhibition of the biosynthetic pathway in vivo shikimate 5 - enol pyruvoyl shikimate -3 - phosphate synthase (5-enolpyruvylshikimate-3-phosphate synthase activity EPSPs). Shikimic acid biosynthetic pathway plants and microbial synthesis of aromatic amino acids in the body, EPSPs is a key enzyme in the process. Death due to glyphosate inhibition of this enzyme, the protein synthesis must aromatic amino acids and related secondary metabolites synthesis is blocked, which interferes with normal biological nitrogen metabolism. Although glyphosate has many advantages of broad-spectrum, high efficiency, but these herbicides kill weeds on crops the same vanishing Health, which limits its scope of application. Therefore, how to overcome the negative effects of glyphosate on crops by the widespread concern, the glyphosate to become a hot research at home and abroad. In order to make full use of the the glyphosate herbicidal advantage, to expand the scope of its use, cultivate glyphosate-resistant transgenic crops is considered to be the most promising application of a weed Countermeasures. At present, the commercial cultivation of glyphosate resistance genetically modified crops are designed for EPSPs, but this mechanism did not Glyphosate degradation. Glyphosate absorbed by the plants may be accumulated in the meristem, reducing crop yields affect plant growth and development. This study found a new glyphosate degradative enzyme gene - glycine oxidase gene, the gene encoded product can be glyphosate degradation into non-toxic compounds, to avoid residues of glyphosate in plants, grasses glyphosate in plants provides a new way for an alternative. In this study, glyphosate contamination Soil DNA construct metagenomic library activity screening methods found positive clones containing the inserted fragment from 110,000 a glyphosate resistant clones, named for the Glp-1. The sequencing results showed that on which contains a glycine oxidase (glycine oxidase, GO) encoding gene, named goA. The gene sequence length is 1110bp, the encoded a 369AA a protein consisting of the theoretical molecular weight of 40686.5Da. Protein homology analysis found that only 88% homology to the ORF encoding product from Bacillus subtilis strain 168 GO. Will goA built into the expression vector pEASY-E1, overexpression in E. coli BL21 (DE3) pLysS. By Ni affinity chromatography and anion exchange chromatography for purification GOA After purification, the specific activity of the GOA 0.283U/mg target protein was 88.9%. Enzymatic assays, GOA able glyphosate degradation of non-toxic compounds carbamoyl phosphate and glyoxylate. GOA right substrates glyphosate, iminodiacetic acid, glycine, and N-ethyl glycine, and so exhibits high catalytic activity. The optimum temperature of the enzyme is 50 ° C, below 50 ℃ for 30min more than 85% of the activity is retained after. The optimum pH of the enzyme is 8.5, and the greater than 85% of the catalytic activity is maintained over a wide pH range (pH7.3-10.3). The the GOA on glyphosate affinity (1/Km) of wild-type GO Pedotti reported 8 times, substrate selectivity of glyphosate (kcat / Km for glyphosate and its glycine kcat / Km ratio) is 5.5 times that of the latter. Molecular dynamics calculations indicate that, the GOA with glyphosate binding free energy of -4.48 kcal / mol GO (-1.90 kcal / mol) of 2.36 times. From simulated GOA / GO and glyphosate complex three-dimensional structure the GOA with glyphosate complex 2 H bonds and H bonds shorter distance. At present, China's glyphosate resistance research is still in its infancy, with the application of the value of glyphosate degradation genes scant. Therefore, it is necessary to mining with independent intellectual property rights of glyphosate degradation genes, and potential application value of genetic resources for the development of resistance to glyphosate or degradation crops.

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