Overexpression Of Cdpk9 Stress Tolerance Of Arabidopsis

CDPK is a kind of Calcium-dependent protein kinases which is first found in plants.It belongs to Ser/Thr protein kinases family,CDPK has a molecular weight of 40～90kDa,Mg2 + works as its assist member。CDPKs are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor.The CDPK family represents diversity and choosable during its signal transduction gateway.Rencently lots of investigations into rice and arabidopsis represent that the CDPK family is comprised of multiplegene family. Arabidopsis includes 12 subfamilies consisted of 34 members. As to rice,27 members. Furthermore most of them involve in adversity signal transduction.we have validated the expression of arabidopsis genes AtCDPK1 and AtCDPK2 are toned up in drought and salt conditions(Urao et al., 1994). In addition, Sheen(1996) make sure that the arabidopsis genes AtCPK10 and AtCPK30 involve in ABA and abiological signal transduction.Thellungiella halophila is close relative to Arabidopsis which is also belong to Cruciferae and has good genetic features such as similar morphology, small genome size, short life cycle, high seed number and an efficient transformation method. However, Thellungiella halophila is able to withstand dramatic salinity shock up to 500mM NaCl. This plant does not produce salt glands or other complex morphological alterations either before or after salt adaptation. Therefore, the study of function of thioredoxin from Thellungiella halophila is quite significant for expatiating plant response mechanism of oxidative stress, understanding the metabolism alteration and adjustment of plant under stresses and expounding the stress tolerance metabolism.The CDPK9 gene was obtained from ESTs (expressed sequence tags) acquired fromλZap-cDNA library of Thellungiella halophila in our lab.Through the expression in vitro and activity experiment we try to prove that CDPK9 did have kinases activity. The family members of CDPK9 exist not only in cytoplasm membrane but also in chloroplast, and different localizations affect their potential functions.By constructing the GFP expression vector of CDPK9 and transforming it into the Arabidopsis anthotaxy, CDPK9 is mostly localized in cytoplasm membrane by laser scanning confocal microscope. Only a few fraction is localized in chloroplast . The subcellular localization results were in accordance with the result of iPSORT software. At the same time ,the CDPK9-GST vector was constructed, amalgamation protein was expressed in DE3 Ecol, Our results showed that the amalgamation protein forms inclusion during its expression. Besides, by constructing the over-expression vector of CDPK9 ,we attained 5 lines of transgene Arabidopsis. and the phenotype of the 5 lines was analyzed. Besides,we also did the Northern bloting experiment,fallaciously,we did not get the hybridize signals on the hybridize membrane.