Clone And Sequence Analyze And Expression Of COR From Descurania Sophia(L.)

Low temperature may hurt plants variously even make them dead. Cold is an environmental factor that limits the geographical distribution and growing season of many plant species. With the development of mechanism of cold-resist at the gene level, some cold-resist genes have been cloned and technology of transgene, it is possible to odtain excellent cold-resist plant through gene-engineering.We use Cruciferae Descurania sophia(L.) as our cold-resist material, which can tolerance to low temperature. Our laboratory successfully cloned the gene cor (cold regulated gene ) from Descurania sophia(L.) . It is useful to clear up the mechanism of cold-resist at the gene level and advance the ability of cold-tolerance. Our research mainly is : 1. Descurania sophia (L.) cor(DsCOR) gene clone. According to homogene in Arabidopsis thaliana and Barassica , a pair of degenerate primers was designed. A EST sequences was obtained by PCR. We use bioinformatics tools analyzing this EST sequence. The result suggest it was high homologous to Arabidopsis thaliana corl5a. The EST sequence is similar to Arabidopsis thaliana corl5a .Then Using the 3' RACE and 5' RACE, we obtain a open reading frame and a complete 3' non-translation sequence of DsCOR . Trought bioinformatics tools analyzing, sequence in the ORF which is 417bp, can express a protein of 139 aa.2. Bioinformatics tools analyzing DsCOR, the similarity to Arabidopsis thaiiana corlSa and Barassica napus BN115 is 85.7 % and 75.4 %. Through software of PHYLIP, we found our obtained gene DsCOR belongs to COR cold-resist gene family.3. Mw of DsCOR is 14.4 kD, pI is 6.83, short of Pro, Cys, Trp. It has the alike second structure with Arabidopsis thaiiana which involves some a—helix second structure. Chlorop prediction results show that it will go to chlorop. signal polypeptide Prediction show that DsCOR has a Precursor polypeptide. And DsCOR has hydrophilic character, then we confer DsCOR may keep soluble in boiling water.4. The characteristic of DsCOR:It has 5 transmembrane regions, which has more transmembrane structure regions than Arabidopsis thaiiana C0R15a. This means DsCOR are more liquid.It can keep membrane steady.Protein site prediction shows that DsCOR has two main site: cAMP- and cGMP-dependent protein kinase phosphorylation site and Protein kinase C phosphorylation site .We infer the two difference between Descurania sophia (L ) and Arabidopsis thaiiana may be the reason why Descurania sophia (L.) have more cold-resisit character than Arabidopsis thaiiana.5. The vector pET32—DsCOR with DsCOR genes was constructed.The gene can well expressed in E. coli BL21 (DS3) . The Mw of fusion protein is 31kD.6. With hydrophilic character, purification the protein in boiling water. A pure protein was obtained.Our experiment is not only important to explain the function of cold resist in Descurania sophia(L.) but also help us to understand phylogenetic analysis in various plants.