Cloning And Construction Of Expression Vector And Transformation Of The Vector Into Arabidopsis Thaliana Of Calcium-Dependent Protein Kinase Gene From Dunaliella Salina

Calcium-Dependent Protein Kinase is a kind of serine/threonine protein kinase, which only exist in plants and some protista. One of the important roles of CDPK is environment stress signal transduction. Specialized expression of CDPK gene and specific accumulation of mRNA will happen under many environmental factor, such as drought stress, salt stress and hypoosmotic. Dunaliella. Salina, enkaryotic unicellular green algae, able to proliferate under saline conditions ranging from 0.1 to 5.5 mol/L NaCl;it was a better mould of studying the salt tolerance mechanism.The reason of the salt tolerance was that there was the gene Calcium-Dependent Protein Kinase in D.Salina.In order to study the salt tolerance mechanism at gene level, this study successfully colned the gene CDPK from D.Salina by the homogene cloning method.Then,the ORF of CDPK was obtained by RT-PCR and was introduced into Arabidopsis thaliana for studying the gene function, the cloning of salt tolerance genes and nurturing salt tolerance plants and sloving the problem of relieving efficient soil salinization .The fellowing is major content:1. Using the homogene cloning methed, we cloned a cDNA fragment of the CDPK from Dunaliella. Salina. The result of DNA sequencing indicated that 60.3% of the sequence of the fragment was homologous to the CDPK in Arabidopsis thaliana. Then, the full-length cDNA of CDPK was constructed by the RACE technique based on this sequence.2. D. Salina was treated with hypoosmotic shock ( NaCl 1.5mol/L～0.5mol/L ) and hyperosmotic shock ( NaCl 1.5mol/L～3.0mol/L ) for 3h, 6h, 12h, 24h, 48h. In order to analyze the effects of various osmotic shocks on the gene expression of CDPK,RT-PCR was performed with an inner standard of 23S RNA from D. Salina. The result showed that hypoosmotic shock and hyperosmotic shock had obviously different effects on the gene expression of CDPK, respectively.3. Transformed to Arabidopsis thaliana by floral dip transformation methed: when inflorescence shoots of Arabidopsis thaliana in flowerpot appeared and the first inflorescence shoot was picked. Second inflorescence shoots were about 6～10 centimeters, immersed in Agrobacterium suspension for 30seconds～6minutes. After three weeks, seeds were harvested. Selection of Kamamycin-resistant showed...