Construction Of Promoter Probe Vector For Acidithiobacillus Ferrooxidans And Isolation And Characterization Of PetⅡ Promoter

Promoter,an essential part in the vector for gene engineering expression,is an important cis-acting element in the regulation of the gene expression.The way of promoter cloning is important for constructing gene engineering vectors and expressing the aimed proteins. In order to research the elements and function of promoter for Acidithiobacillus ferrooxidans,site-directed mutagenesis was used to create a new BstBI enzyme recognize site in pSV-β-galactosidase vector, of which was the backbone.Resulted promoter probe vector pSVB could replicate in Escherichia coli.The petⅡoperon was constituted with cycA2,sdrA2,petA2B2C2.It could encode a bclcomplex.A putative promoter region,the nucleotide upon the cycA2 gene from A.ferrooxidans was isolated by PCR;gpt promoter of the promoter probe vector pSVB was replaced by this DNA fragment.Resulted plasmid named as pAP2.After transform E.coli,The activity of this promoter region was determined by detection of theβ-galactosidase activity in the host cells.The result of reporter gene assay showed that the DNA fragment upon the cycA2 gene from A. ferrooxidans exhibited a distinct promoter activity.This vector could be used to clone and identify potential promoter region of some important functional genes from A.ferrooxidans.Sequences analysis for petⅡpromoter region showed that there were two putative core element -35 and -10 box,which were "TTGATA" (-95bp to -90bp) and "TATGCT"(-59bp to -54bp).A single-base pair-mutagenesis,which just the first base "T" to "G" was carried out in -95bp to -90bp region,and "T" to "C" in -59bp to -54bp region, respectively.The results of reporter gene assay showed that two mutations decreasedβ- Galactosidease activity to 15%and 59%of the wild-type respectively.It suggested that these two region were important for petⅡpromoter activity,they were the -35 box and -10 box sequence,respectively,of the petⅡpromoter.