| Heat Shock Protein (HSP) is a protection protein that is induced in response to environmental stress byregulate relate proteins, its distribute in various species. HSP70as the main molecular chaperone, wasstudied in the molecular level of gene and protein deeply. Viola tricolor is a potted flowers that it is lesstolerant of heat, as the main ornamental flower which has rich color and long flowering, the thermolabilewas serious limited its application.To obtain the HSP70gene and analysis its characteristic has greatsignificances to study the thermotolerance and thermotolerant germplasm breeding in the Viola tricolor.The Viola tricolor HAR was heat-treatment20min，40min and60min at42℃, to induce the HSP70gene expressed. Using blast software to search for the HSP70gene of plants which have close relationshipwith Viola based on the homology between Arabidopsis thaliana, Gossypium hirsutum and other palntHSP70gene in GenBank, analyzed their conservative structures, then got the homologous gene as themodel to designed two pairs of primers and the VtHSP70gene was amplified applied the sequencehomologous cloning method, the length of the VtHSP70gene was882bp. The residual5’ends and3’endssequences were cloned by the RACE technology, the complete2169bp of HSP70gene was cloned based onthe three fragments. One fragment was cloned into the pMD18-T vector and we got the recombinantcloning vector HSP70-pMD18-T.The VtHSP70gene sequence was analyzed and predicted by bioinformatic tools. The result showedthat the length of Open Reading Frame was1950bp, coded649amino acids. In the analyzed ofphylogenetic tree, the HAR nucleotide sequence of CDS shared47%homology with theHSP70gene withthe Brassica napus and Thellungiella halophila, belong to the closer genetic relationship species. Theprediction of HSP70showed thatHSP70was hydrophobic protein existed in the outside membrane, no signal peptide, belong to NBD-sugar-kinase-HSP70-Actin superfamily, have the same similar sitewithHSP70-1, HSP70-2andHSP70-3. |
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