[Objective] The aims were to obtain cloning of HDR gene from Ginkgo biloba.and study its function.[Method] The coding sequence of HDR gene was cloned from G.biloba by reversed transcription polymerase chain reaction,w...[Objective] The aims were to obtain cloning of HDR gene from Ginkgo biloba.and study its function.[Method] The coding sequence of HDR gene was cloned from G.biloba by reversed transcription polymerase chain reaction,which was designated as GbHDR （GenBank accession No.：DQ364231）.The cDNA full-length of GbHDR is 1 827 bp containing a 1 425-bp open reading frame （ORF） encoding a 474-amino-acid polypeptide and constructed into the prokaryotic expression vector pTrcGbHDR.The β-carotene biosynthetic pathway in E.coli strain XL1-Blue was reconstructed by transforming with pAC-BETA.This engineered XL1-Blue was transformed with pTrcGbHDR.[Result] A 1 441 bp GbHDR was obtained containing a 1 425-bp ORF encoding a 474-amino-acid residues of protein,the predicted molecular weight was 53.2 kD,and predicted isoelectric point was 5.76.Functional complementation assay indicated that GbHDR could promote theβ-carotene accumulation in engineered XL1-Blue harboring pTrcGbHDR and pAC-BETA,and as a result,the engineered bacteria showed the brightly orange given by β-carotene.This suggested that GbHDR had the typical function of known HDR genes.[Conclusion] A engineered bacteria of E.coli which could highly accumulate β-carotene was obtained,which will provide candidate genes and targets for realizing β-carotene metabolic engineering.展开更多
基金Supported by National Natural Science Foundation (30500303)~~
文摘[Objective] The aims were to obtain cloning of HDR gene from Ginkgo biloba.and study its function.[Method] The coding sequence of HDR gene was cloned from G.biloba by reversed transcription polymerase chain reaction,which was designated as GbHDR （GenBank accession No.：DQ364231）.The cDNA full-length of GbHDR is 1 827 bp containing a 1 425-bp open reading frame （ORF） encoding a 474-amino-acid polypeptide and constructed into the prokaryotic expression vector pTrcGbHDR.The β-carotene biosynthetic pathway in E.coli strain XL1-Blue was reconstructed by transforming with pAC-BETA.This engineered XL1-Blue was transformed with pTrcGbHDR.[Result] A 1 441 bp GbHDR was obtained containing a 1 425-bp ORF encoding a 474-amino-acid residues of protein,the predicted molecular weight was 53.2 kD,and predicted isoelectric point was 5.76.Functional complementation assay indicated that GbHDR could promote theβ-carotene accumulation in engineered XL1-Blue harboring pTrcGbHDR and pAC-BETA,and as a result,the engineered bacteria showed the brightly orange given by β-carotene.This suggested that GbHDR had the typical function of known HDR genes.[Conclusion] A engineered bacteria of E.coli which could highly accumulate β-carotene was obtained,which will provide candidate genes and targets for realizing β-carotene metabolic engineering.
