Cloning Of Two Small Heat Shock Proteins From Mirabilis Jalapa L. And Transformation Of Its Fusion-gene Into Saccharomyces Cerevisiae

Due to the rapid development of the economy and increased pace of of petroleum extraction,the resulting problem of pollution is increasingly significant. In recent years,phytoremediation is widely used in the repair of petroleum contaminated soil,when it comes to this issue,a question that deserves our attention is how the plants cope with the abiotic stresses imposed by polyaromatic hydrocarbon and other organic pollutants. The previous comparative proteomic data shows that under the stress of petroleum,the expression of two Hsps(Hsp18.2 and Hsp17.7) was remarkably upregulated compared to the control group.In this article,the ORF of the two Hsps described above is cloned using RACE(Rapid amplification of cDNA ends).By fusion PCR method,Hspl8.2 and Hsp17.7 was linked with 2A peptide for the purpose of balance expression of the two proteins.Finally.the three ORFs was cloned into pYES2 vector and transformed into saccharomyces cerevisiaeo in the hope of conjecturing the roles these Hsps played in the stress tolerance formation of mirabilis jalapa.The research results is listed as follows:1. The complete ORFs of Hsp 18.2(483 bp) and Hsp 17.7(474 bp) was obtained by RACE,which encodes 161 and 158 amino acid residues and weight about 18.2 kDa and 17.7 kDa respectively,both of them owns an conservative domain named ACD that is typical of small heat shock proteins.2. Using 2A peptide and fusion PCR method.a fusion intracellular ORF was obtained,which can express the two proteins(Hsp18.2-Flag and Hspl7.7-HA) simultaneously and evenly by a single ORF.3. Three recombinant yeast expression vectors(pYES2-HSP18.2-Flag、pYES2-HSP17.7-HA and pYES2-HSP-fusion)was constructed,each of them was verified by double digestion and sequencing.4. Successfully transformed the recombinant yeast expression vectors described above into saccharomyces cerevisiaeo and screened out positive clones.