Isolation, Expression Analysis And Interaction Analysis Of OsHsp82

Heat Shock Protein(Hsp) is a kind of protein that accumulate under heat shock condition and it is important to the response under stress conditions and the development of the plant.The Hsp90 family is a class of Hsp whose molecular weight is varied from 80kD to 90kD.It helps a lot in the folding and maintaining the conformation of proteins.Hsp90 is evolutionarily conserved,and the similarity of Hsp90 in eukaryote is more than 50%.The Hsp90 is divided into three domains, which is the N-terminal domain,the middle charged domain and the C-terminal domain.The N-terminal domain is the site of ATP binding and hydrolysis,and the C-terminal domain which is ended with the MEEVD sequence provides a strong dimerization interface.Different kind of proteins are recognized and bound by the three domains of Hsp90.Nowaday the research of Hsp90 is mainly focused on mammalian.More and more interaction proteins of Hsp90 have been reported by researchers according to their study in mammalian,but the study of plant Hsp90 is limited.To understand the function of Hsp90 under stresses,we isolated the OsHsp82 gene,and we analyzed its expression under heat and cold circumstance and we studied the interaction protein of Hsp90 by using H2Y.The major results are showed below.1.Using Fluorescent Differential Display(FDD) method,the mRNA expression in rice leaves and roots under low temperature and drought stress was compared to normal growth condition.One interest fragment was isolated by combining the H.A.Yellow-PAGE(contained 0.1%H.A. Yellow) separation and Macroarry Screening methods.The fragment shows more than 95% similarity of OsHsp82 cDNA,and we isolation the full cDNA sequence according to the information of GenBank.At the amino-acid sequence level,OsHsp82 exhibit high homology to ZmHsp82,AtHsp90-2 and HvHsp90(84.9%,90.7%and 94.7%respectively),and all of them end with the MEEVD sequence.2.By simi-quantitative RT-PCR analysis,the expression of OsHsp82 at the transcription level was both increased under the high and low temperature.Especially under high temperature,the expression level of OsHsp82 increased more sharply and vigorously.Those results represent OsHsp82 is take part in the response to high and low temperature of rice.3.Using OsHsp82 as a bait protein,we isolated 7 cDNA of interaction protein from the cDNA pool constructed by the rice under the drought and cold condition.The deduced proteins are PRP1,RBBI3-3,PSâ…¡10kD peptide,CYP98 and 3 unknown protein.We also discussed the function,possible binding model and the regular function of the compound.4.We constructed a pGEX-Hsp82 vector.And we also expressed and purified the OsHsp82 in vitro.Meanwhile,the pET-RBBI3-3 vector was constructed and the RBBI3-3 was expressed in vitro.But unfortunately,the interaction between OsHsp82 and RBBI3-3 was not detected in vitro.The possible reason has been discussed.