Characterization, Expression Analysis Under Salinity Stress Of Calcium-Dependent Protein Kinase Genes In Rice And Genetic Transformation Of Two Putative Important CDPK Genes

Calcium-dependent protein kinases(CDPKs)are classified into one kind of Ser/Thr kinases in plants.CDPKs play an important role in mediating the signal transductions of growth and development,and the abiotic stresses.Update,it is still in lack of a thoroughly elucidation for the CDPKs in rice.Therefore,in total 22 CDPK genes of rice were identified by searching the rice genome sequences which were released in the GenBank in this study.Based on the approaches of modern molecular biology and bioinformatics,the gene structures of the rice CDPK genes,the expression patterns of the rice CDPK genes under salt stress condition,and the functions of the promoter and gene of several tested rice CDPK genes were studied.The main results were as follows:1.There were huge differences on the structure properties among the rice 22 CDPK genes.The DNA length,cDNA full length,open reading frame length,and the translated amino acid numbers among the rice 22 CDPK genes were dramatically diverse.It could be classified into two sub-groups for the rice 22 CDPK genes based on phylogenetic tree analysis.2.Under normal growth condition and salt stress condition,some of the rice CDPK genes showed an expression pattern with a constitutive type,including OsCDPK4, OsCDPK18,OsCDPK19 and OsCDPK24.The transcriptions of OsCDPK10 and OsCDPK16 were regulated specifically by salt in the leaves and roots.The expression of OsCDPK6,OsCDPK20 and OsCDPK13 were specifically up-regulated in leaves under salt stress condition.Meanwhile,the time-course changes of the transcripts of OsCDPK6 and OsCDPK20 were a curve line with a one peak,at the time point of 1h after salt treatment. Therefore,it is indicated that many rice CDPK genes are involved in the salt signal transduction.3.Based on the cloning of the OsCDPK19 promoter,the binary vector pCAM3301-OsCDPK19 fused the OsCDPK19 promoter in front of the reporter gene GUS (β-glucuronidase)were constructed.It is found that the OsCDPK19 promoter could drive the GUS gene to be transcribed at a high level,with an expression pattern of the position of vascular tissue predominately.The study indicated that there were several enhancers to be located at the fragments of -296 bp～-623 bp and -623 bp～-989 bp.The AC-Ⅱ-like motif and AC-Ⅲ-like motif located at the fragment of -989 bp～-1474 bp could possibly be related to the reporter gene with an expression pattern of predominately in vascular tissue.4.The dramatically induction of OsCDPK6 in leaves under salt stress condition indicated its involvement in the salt signal transduction.Thus,the tobacco transgenic lines with the overexpression of OsCDPK6 were generated based on the Agrobacterium-tumeficiens mediated genetic transformation approach.Under salt stress condition,the salt tolerance in the transgenic lines with up-regulated OsCDPK6 expression was obviously improved compared with the control.After 7d salt treatment,there were less injury and much more leaf area per plant in the transgenic lines.Meanwhile,compared to the control,the superoxide dismutase(SOD)activities were higher and the malonaldehyde(MDA)contents were lower in the transgenic lines.The contents of chlorophyll a,chlorophyll b,carotenoid,soluble sugar,and soluble protein were also higher than the control.Taken together,the cellular protection metabolic pathway was response to the salt signal transduction mediated by OsCDPK6.5.From the induction of OsCDPK6 in the rice leaves under salt stress condition,it is suggested that the promoter of OsCDPK6 has the capability to drive the downstream gene to be expressed with a pattern of up-regulation when imposed to the salt.So this promoter perhaps has a value for establishment of the novel crop cultivars with high level of salt tolerance by biotechnology.Therefore,the function of the OsCDPK6 promoter was evaluated by fusing this promoter into the tobacco plants based on the cloning and genetic transformation techniques.The results indicated that the histochemical staining intensity of the root,and leaf was accordance to the expression pattern of OsCDPK6 in rice,in the two conditions of normal growth or salt stress.Under the salt stress condition,the staining of stem and leaf were obviously intensified than that under normal growth condition.The expression pattern of the reporter gene was constitutive under the control of OsCDPK6 promoter.Based on the identification of PLACE analysis,one recognition motif of transcription factor MYC and three core recognition motif of transcription factor MYB were identified in the OsCDPK6 promoter.So,the induced expression of OsCDPK6 in the leaves under the salt stress condition was possibly involved in the pathway in which ABA activated the transcription factor MYC and MYB or initiate novel MYC and MYB synthesis.