Fundamental Function Analysis Of HSP90?HSC70 And HSP70 Gene In Haliotis Diversicolor

Heat shock proteins(HSPs)are highly conserved proteins which are generated on the condition of high temperature and other stresses.HSPs,known as molecular chaperones,which are existed from prokaryotic cells to eukaryotic cells,play important roles in the folding,translocation,refolding and degradation of proteins.HSP70 and HSP90 are the most important members of HSPs family which play key roles in the response to different stresses and have the function of resistance to apoptosis and cell immune.Promoter is located in the 5' flanking region of DNA and can be detected and combined by RNA polymerase.Promoter,as an important ciselement in regulating gene expression,determines the activity of the gene,so its function is becoming a research hotspot.In this study,on the basis of the sequence of Haliotis diversicolor,HdHSP90 cDNA cloned by our previous study and the sequence of HdHSP70 and HdHSC70 published in NCBI,the 5' upstream regulatory sequences of the three HSPs were cloned by genome walker,Tail-PCR and conventional PCR.Meanwhile,the construction of recombinant plasmids,cell culture,transient transfection and dual-luciferase reporter assays were also used to analyze the transcriptional regulation of HdHSP90,HdHSP70 and HdHSC70 at transcriptional levels.The results are listed as follows:1)2800 bp,2013 bp and 2140 bp of 5' flanking region of HdHSP90,HdHSC70 and HdHSP70 were obtained respectively by the genome walker and Tail-PCR.2)Bioinformatics analysis showed that there were a TATA box in the upstream-30—-32 bp of the predicted transcription start site(A)and a CpG island in the different position in the three HSP genes.Also,the three had a lot of common transcription factor binding sites,such as ATF,NF-1,TBP,Sp1,Oct-1,C/EBPalp and so on,and some heat shock elements.3)The core promoter regions of HdHSP90,HdHSC70 and HdHSP70 were located between-98 bp and +83 bp,between-104 bp and +43 bp,and between-189 bp and +46 bp,respectively.4)All seven constructed plasmids with different deletion fragments of HdHSP90 had high activity.Compared with other plasmids,activity of pGL-90-6r was the highest.The analysis of mutant transcription factor binding sites between-624 bp and-539 bp indicated that Oct-1,C/EBPalpha and NF-1 can inhibit the HdHSP90 gene expression in a certain extent.5)All seven constructed plasmids with different deletion fragments of HdHSC70 also had high activity,but the activity of pGL-c70-7r was lower than that of pGL-c70-6r(P < 0.01).Mutation analysis of transcription factor binding sites between the two shortest deletion fragments found that the ICSBP close to the position of-252 bp can enhance the expression of HdHSC70 gene.6)Activities of seven constructed plasmids with different deletion fragments of HdHSP70 promoter were also high.Among them,the activity of pGL-70-2r was the highest and was significantly(P<0.05)higher than those of pGL-70-1r and pGL-70-3r.However,the relative fluorescence activity of the seven constructed plasmids with deletion fragments of HdHSP70 gene is lower than that of HdHSC70 gene.