| Heat shock proteins(HSPs),a family of highly conserved proteins with molecular mass from 15 to 110kDa,are ubiquitous,occurring in all organisms from bacteria and yeast to humans.They are present in the cytosol,motochondria,endoplasmic reticulum,amd nucleus within cells.The expression of HSPs is induced by a range of stressor,such as high temperature,low temperature, radiation,heavy metals,bacterial and viral infection,sensceine,inbreeding.HSP70 is the most temperature sensitive and highly conserved family member of the HSPs.Also HSP70 are the most widely studied stress proteins and play important physiological roles in normal conditions and situations involving both systemic and cellular stress.HSP70 assist a wide range of folding processes,including the folding and assembly of newly synthesized proteins,refolding of misfolded and aggregated proteins,membrane translocation of organellar and secretory proteins,and control of the activity of regulatory proteins.In addition,HSP70 demonstrate its potential protection against environmental stressors.However,the molecular mechanism of the planarians' distribution and their life habits has not been reported.So the research on planarians HSP70 gene and protein is very impotant for solving the relationship between planarians and environmental adaptation.The planarian Dugesia japonica is the representative species of the phylum Platyhelminthes, Tricladida,and generally found in nature in the overcast cool freshwater.Currently,the mass of the research on the planarians is about the machanism of regeneration and molecular biology.There is no report on molecular mechanisms about planarians adaptation to their habit.So studies on interaction between HSP70 and environmental adaptation will be very important.The full-length cDNA of D.japonica HSP70-1 and HSP70-2 was cloned by RT-PCR and RACE methods and deduced amino acid sequence was analyzed by bioinformatics.The results of investigations are offered as follows:1.The full-length cDNA of HSP70-1 is 2118bp,containing a 5'-untraslated region(5'-UTR) of 59bp,a 3'-UTR of 115bp and a ployadenylation signal(AAAAAA).The content of GC is 44.9%.The open reading frame(ORF) of HSP70-1 is 1944bp,which encodes a deduced 647 amino acid peptide with a predicted molecular mass of 71.0kDa and with the theoretical pI of 5.55.2.The full-length cDNA of HSP70-2 is 2139bp,containing a 5'-UTR of 39bp.a 3'-UTR of 156bp and a ployadenylation signal(AATAAA).The content of GC is 36.1%.The ORF of HSP70-2 is 1941bp,encodes a deduced 646 amino acid peptide with a predicted molecular mass of 71.3kDa, and the theoretical pI is 5.37. 3.Sequence analysis showed that,HSP70-1 and HSP70-2 are both hydrophilic proteins,and mainly locates in the cytosol.A HSP70 9-element fingerprint,3 HSP70 signatures,2 nuclear localization signals,the ATP-GTP-binding site,the non-organellar consensus motif,the cytoplasmic HSP70 carboxyl terminal region were found in HSP70-1 and HSP70-2.Analysis of protein secondary structure prediction showed that,HSP70-1 and HSP70-2 are composed of N-terminal highly conserved ATPase domain and C-terminal lightly conserved peptide-binding domain.4.The results of sequence alignments analysis by ClustalX showed that two protein share 73 96.6%identity with the other known HSP70 in animals.The similarity of HSP70 amino acid sequences in different species suggests similar molecular functions.The phylogenetic tree constructed by NJ method showed that,HSP70-1 and HSP70-2 clustered first with other platyhelminthes HSP70 on the tree,and then with other HSP70 from vertebrates,arthropoda,insects, and nematode.This work is the first report about D.japonica HSP70.It has information for HSPs research on D.japonica.Combined with previous studies on the results of HSP70,it is believed that HSP70 play a very important role in the planarian adapting to changes in ambient temperature,and laying the foundation for further molecular biology research on interaction between HSP70 and environmental adaptation in D.japonica. |
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