Functional Characterization Of Gene Cphsp1

The heat shock protein (HSP) are an evolutionarily conserved family of proteins synthesized by organism in response to adverse stress. Heat shock proteins are expressed when cells exposed to elevated temperatures, or to other kinds of environmental stress. Heat shock proteins mainly take part in transporting, folding, assembling, mapping of new peptides as well as renaturation and degradation of denaturated proteins in organisms. These protein s increased expression canprotect the organism against stress-induced damage. Heat shock proteins are among the most abundant intracellular proteins. According to the molecular size, heat shock protein divided into five groups:HSP100,HSP90,HSP70,HSP60 and the small heat shock protein.Based on the previous studies in this laboratory, in this study,we have cloned Chimonanthus Praecox heat shock protein gene, named Cphspl. We have finished the construction of the plant expression vector through Agrobacterium-mediated transformation of Nicotianatabacum, obtaining T1-generation transgenic plants. The main findings are follows:1. Cloning of CphsplWe have found the cDNA sequence with the size of the 447bp, by rough randomly selection from cDNA library sequencing. Sequence analysis showed that the gene encode 158 amino acids.BLASTX comparison showed that the heat shock proteins homology higher prima facie by the heat shock proteins of the cDNA encoding heat shock proteins,named Cphspl.2. Construction of Plant Expression VectorCphspl gene is in the cDNA library cloning vector pTriplEx2, activate bacterial liquid respectively including pTriplEx2 and pMD-18TM and extract plasmid, then digest plasmid by restriction endonuclease SacI and XbalI. Recycle target gene and then link to cloning vector pMD-18TM. The recombinant plasmid pMD-18TM-Cphspl is successfully constructed and confirmed by restriction endonuclease digestion and PCR.Digest pMD-18TM-Cphspl by double restriction endonuclease XbaI, SacI, then recycle the target gene and link to expression vector pC2301.The identification of PCR and enzyme cutting show that the construction of the recombinant pC2301-CpEXP2 plasmid could be confirmed. 3. The genetic transformation of Arabidopsis thalianaWe transplanted the Chimonanthus Praecox heat shock proteins gene Cphspl into Nicotianatabacum by Agrobacterium tumefaciens. Through the Km+ resistance screening detection and PCR amplification. It proves that the initial Cphspl gene has been integrated into the Arabidopsis genome primarily and transgenic plants is growing normally.4. Abiotic stresses respond analysis of Cpshpl from Chimonanthus praecox (L.) Link by real time quantitative PCR.The Chimonanthus praecox (L.) Link plants were treated by cold temperature and hot temperature. Then real-time quantitative RT-PCR was used to analysis the expression of Cphspl in Chimonanthus praecox (L.) Link leaves. The results revealed that the expression of Cphspl were differently regulated by cold and hot.. In addition, it was found that it was induced by hot temperature and had high-level expression.