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Molecular Cloning And Characterization Of Dihydrolipoamide Dehydrogenase Gene In The Silkworm, Bombyx Mori

Posted on:2011-05-28Degree:MasterType:Thesis
Country:ChinaCandidate:J HuoFull Text:PDF
GTID:2120360302493736Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
Dihydrolipoamide dehydrogenase(DLDH),a flavin-dependent oxidoreductase,is an ubiquitous enzyme which presents in variety of organisms and plays an important role in energy metabolism.To date, DLDH have been identified in many species,however,no reports have been published on DLDH in Bombyx mori(BmDLDH).In this study,we obtained the BmDLDH cDNA sequence from NCBI and performed both bioinformatics and experiment analysis on BmDLDH gene.The results showed that BmDLDH gene contained 11 exons and 10 introns,and the cDNA of the gene contained an open reading frame(ORF) of 1491 bp,encoding a protein of 496 amino acid residues with theoretical molecular mass of 52.9 kDa,theoretical isoelectric point of 8.8.There is a mitochodria targeting peptide(MTP) composed of 27 amino acids at the N-terminal of the protein.In the present study,the putative gene was cloned,expressed in E.coli BL21(DE3) and purified by metal chelating affinity chromatography under both nature and denature conditions.The amino acids sequence of recombinant protein was confirmed by mass spectrographic analysis and the purified recombinant protein was used to analyze enzyme activity and to raise polyclonal antibody against BmDLDH for subcellular localization study and protein expression pattern analysis.Reverse transcription polymerase chain reaction(RT-PCR) analysis of DLDH in Bombyx mori showed the gene was expressed in all developmental stages and various fifth instarlarvae tissues of silkworm.Western blotting analysis of BmDLDH in different tissues from the fifth instar larvae confirmed that DLDH was expressed in various larvae tissues.Immunofluorescence and subcellular fractionation studies of BmN cells showed that majority of BmDLDH proteins were localized to the mitochondria.DLDH catalyzes the interconversion of dihylipoamide and lipoamide with concomitant interconversion of NADH and NAD~+.Based on this feature,the activity of the enzyme was measured.The result of activity assay indicated that the recombinant protein expressed in soluble form was able to catalyze the NAD~+-dependent oxidation of lipoamide in the reverse reaction.
Keywords/Search Tags:Bombyx mori, dihydrolipoamide dehydrogenase, bioinformatics analysis, RT-PCR, enzymatic activity, subcellular localization
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