| Calcium dependent protein kinases (CDPKs) are a kind of serine/threonine protein kinases that are only present in plants and some protist. The activity of CDPKs is dependent on Ca2+ but not on calmodulin. CDPKs have a molecular mass of approximately 40～90kDa. CDPKs are a multigene family and there are 34 CDPKs in Arabidopsis thaliana and 31 CDPKs in rice.As a second messenger in plant cells, Ca2+ plays a key role in regulating the processes from perceiving environmental stress signals to down-stream gene expression. The activity of CDPKs is regulated by calcium ions, reversible phosphorylation, phospholipids and 14-3-3 proteins. Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways in plants. CDPKs have many different substrates, which reflect the diversity of their functions. Potential protein substrates of CDPK are involved in carbon and nitrogen metabolism, phospholipid synthesis, ion and water transport, cytoskeleton organization, transcription and hormone responses, growth and development regulation, biotic and abiotic stress responses in plants. Some study shows that CDPKs of Arabidopsis thaliana are distributed in endoplasmic reticulum (ER), peroxisome, cell membrane, cytoplasm, nucleus, mitochondria.As a model plant, Arabidopsis thaliana has lots of merits. But Arabidopsis thaliana is a typical glycophyte, and its tolerance to salt is low. Thellungiella halophila, which belongs to Cruciferae, is a close relative to Arabidopsis 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 500 mM NaCl.The CDPK9 gene was obtained from ESTs (expressed sequence tags) acquired fromλZap-cDNA library of Thellungiella halophila in our lab. This project aims to study the function of ThCDPK9 gene. The full length of this gene is 1614 bp, and the protein encoded by this gene is constitutive of 537 amino acids (about 60 kDa). iPSORT software prediction indicated that ThCDPK9 has no signal peptide sequence and is not mitochondria or chloroplast protein.In order to understand the biological function of ThCDPK9 gene, we firstly construct plant ThCDPK9-EGFP binary expression vector. This construct was transformed into the onion epidermal cells by agrobacterium-mediated transient transformation, but we do not detect the GFP signal. To further verify the result, this construct was transformed into the Arabidopsis by flora dipping and obtained 30 lines. At the same time, we constructed GST-ThCDPK9 vector and transducted into E.Coli strain BL21(DE3). The BL21 cell grew for 4 h at 28℃after 0.8 mM IPTG induction, the GST-ThCDPK9 amalgamation protein was Soluble and existed in supernatent. The purified protein can be used to protein kinase activity assay and other studies.We construct gene silencing vector of ThCDPK9 and this construct has been transferred into Agrobacterium tumefaciens GV3101. Thellungiella halophila was transformed with resultant GV3101 by floral-dip method, and T0 transformants were screened with Basta. Finally, we obtained 25 Basta-resistant lines. The molecular identifications of these basta-resistant plants are as below:1. PCR result showed that all basta-resistant plants had strong positive signals, and no signal was shown in wild type plants.2. RT-PCR result showed that we have obtained two lines where the level of RNA expression is weaker than in wide type.
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