Dataset Information

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori.

ABSTRACT: Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.

SUBMITTER: Wang N

PROVIDER: S-EPMC3115317 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori.

Wang Nan N Shi Haifeng H Yao Qin Q Zhou Yang Y Kang Lequn L Chen Huiqin H Chen Keping K

Genetics and molecular biology 20110401 2

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH p ...[more]

PMID: 21734824

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Project description:BACKGROUND: Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. RESULTS: Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. CONCLUSION: B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically expressed in midgut, integument and head, implying that these genes may have important roles in detoxifying secondary metabolites of mulberry leaves, contaminants in diet, and odorants. Our results provide some new insights into functions and evolutionary characteristics of COEs in phytophagous insects.

Dataset Information

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori.

Publications

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori.

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