Cloning And Functional Analysis Of CiCPK32 From Caragana Intermedia

Calcium dependent protein kinases(CDPKs/CPKs)is widely found iin plants and protists.As receptor and effector proteins of Ca2+,CPKs play a key role in signal transduction.Moreover,CPKs are essential in plant normal growth and resistance to adversity.Caragana intermedia is a good afforestation tree species in desertification areas of north China,which is resistant to drought,cold,high tenperature,salt and alkali.In this study,Caragana intermedia was used as a material to clone a CiCPK32 gene by RACE technology,and its primary functions under different stress conditions were studied.The results are as follows:1.The cDNA of the CiCPK32 was 2074bp in length,the open reading frame was 1566bp long,encoding 521 amino acids,and the molecular weight of the predicted protein was 58.91 kD.Amino acid sequence alignment and phylogenetic analysis showed that the protein had 77%homology with CPK32(AT3G53040)in Arabidopsis thaliana,so the gene was named CiCPK32.2.Real-time fluorescence quantitative PCR was used to detect the expression of CiCPK32 gene under different stress conditions,and the results showed that it was induced by drought,ABA and hypersaline treatment,suggesting that it gene might be related to the response mechanism of C intermedia to stress.3.The overexpression vector p35s::CiCPK32-HA was constructed and was transformed into wild Arabidopsis thaliana.The expression of CiCPK32 in the homozygous lines of transgenic Arabidopsis thaliana increased in various degrees.4.Under the treatments of 400 mM mannitol,100 mM NaCl and 0.4 ?M ABA,the germination rate of CiCPK32 overexpression lines was higher than that of the control.The primary root ofthe transgenic seedlings under 12 mM ABA treatment was longer than that of the control.5.The water loss rate of the 3 week-old CiCPK32 over-expressed lines was lower than that of the control,and showed a significantly higher survival rate than the control under drought treatment.6.Under 100 ?M ABA treatment,the transcription level of stress-related genes were up-regulated in all over-expressed lines compared to the control.To sum up,CiCPK32 positively regulates the tolerance of the transgenic Arabidopsis to drought and salt stress.