Cloning And Expression Analysis Of Tomato HSP108.6,HSP60α/β Subunit Gene

All plant, animal, inset, bacterial and fungal species so far examined produce a new set ofproteins (heat shock proteins) in response to temperatures above optimum. These proteins functionas molecular chaperones to prevent aggregation of denatured proteins, to assist in folding of nascentpolypeptides, to aid in folding of nascentor to resolulize aggregated denatured proteinsSeveral classes of Hsps have been described including Hsp100, Hsp90, Hsp70, Hsp60, Hsp40,and the small Hsps.The Hsp100/ClpB proteins are believed to be involved in the ATP-dependentdissolution of cytosolic or nuclear protein aggregates formed during heat stress. Roles for chloroplastcpn60's have been suggested, including: general chloroplast protein import,chloroplastdifferentiation , fruit ripening .In this study, five super long genes were cloned from the cDNA library prepared by heat shockflowers of tomoto through EST analysis, which are chaperonin-60 alpha subunit;chaperonin-60 betasubunit;ClpB heat shock protein ;TPR-repeat protein ; heat shock protein 83;peptidylprolylisomerase.The cDNA of chlorolplast HSP108.6/ClpB gene was 3144 nucleotides long encoding for 984amino acids . The analysis of characteristic sequence and homology of ClpB indicated that it had ahigh amino acid sequence identity with Arabidopsis thaliana Hsp109 and Phaseolus lunatuschlorolplast ClpB , which present a purative chlorolplast transit sequence.Antibody was prepared byimmune rabbit with GST-cp HSP108.6 protein. We demonstrate that it imports to chloroplast by theWesthern blot first time. Southern blot analysis showed that there are was more than one copy ofHSP108.6 in tomato genome. Northern blot analysis indicated that the transcription of HSP108.6was obviously up-regulated by heat shock in leaf cells and its transcription increase at 32℃.Theresults of Western blot were similar with Northern blot. The expression of HSP108.6 wasconstitutive demonstrated by Western blot in tomato fruit and accumulates increased with increasedtemperature .The cDNA of chlorolplast Hsp60 αsubunit gene was 2117 nucleotides long encoding for 579amino acids, Hsp60 βsubnit was 2207 nucleotides long encoding for 509 amino acids. Antibodyagainst Hsp60 βsubunit were prepared by immune rabbit with GST fusion protein .The differences of the expression of chlorolplast HSP108.6 and Hsp60 were compared ofamong chlorolplast HSP108.6,Hsp60 and sHSP. The results indicated that the expression of Hsp60was constitutive, while the expression of chlorolplast HSP108.6 and sHSP was heat-induced. Thethreshold tempreture of chlorolplast HSP108.6 expression was 32℃, while that of sHsSPexpression was 36℃. So there were different expression patterns in heat shock protein famly.