Gene Cloning And Analysis Of Calcium-dependent Protein Kinases In Nicotiana Tabacum

Calcium-dependent protein kinases (CDPKs) are essential sensor-transducer of calcium signalingpathways in plants. Functional characterization of CDPKs is of great interest because they playimportant roles during growth，development，and in response to a wide range of environmental stimuli.Therefore，this study mainly focused on two aspects: On the one hand，CDPK genes were furtherisolated from common tobacco (Nicotiana tabacum) by rapid amplification of cDNA ends (RACE)，andanalyzed their sequences characteristics，evolutionary relationships and gene expression. On the otherhand，obtaineded the transgenic RNAi plants of the CDPK genes，and preliminary explored andanalysed the genes related functions by using the plants. In this study，the main results were as follows:(1) Four genes termed NtCDPK13、NtCDPK14、NtCDPK15and NtCDPK16(GenBank AccessionNo. JN662018、JN662019、JN662020and JN662021) were separated by using the RACE method.Multiple sequence alignments showed that all the protein sequences encoded by the four genes containthe typical CDPK-conserved domains，which had well-conserved C-terminal calmodulin-like structurewith4-EF hand motifs for calcium-binding. Phylogenetic analysis indicated that the NtCDPK13、NtCDPK14and NtCDPK16might be the specific genes in Nicotiana tabacum，but the NtCDPK15might originate from Nicotiana tomentosiformis.Last，forecasted paralogous genes NtCDPK15andNtCDPK1might correspond to conserved functions.(2) The results of real-time quantitative reverse transcription-PCR (qRT-PCR) showed that theexpression patterns of the four genes were significantly different. The expression of NtCDPK13couldnot be induced by high-salt，drought or ABA treatment. The NtCDPK14highly expressed in roots undersalt stress. The expression of NtCDPK15highly increased in stems and leaves when treated withhigh-salt，and ABA treatment also made the gene expression increased in roots. The NtCDPK16expression significantly increased in roots under the salt，drough and ABA stress.(3) Constructed the NtCDPK14and NtCDPK16RNAi expression vectors by using the Gatewaycloning technology.Via agrobacterium-mediated tobacco transformation methods，acquired the twogenes RNAi plants. The result of hygromycin resistance detection confirmed that the transformationratio was above70%. After further cultured，acquired T1generations. The results of both hygromycinand targent fragments PCR amplification at DNA level showed that the transformation ratio of T1transgenic lines was between75%and85%. Real-time quantitative showed that in the RNAi lines therelative expression of NtCDPK14and NtCDPK16were significantly decreased in roots and leavescompared with the wild-type，this confirmed the two genes were really interferenced.(4) According to the preliminary analysis of the RNAi lines，it is suggested that NtCDPK14andNtCDPK16might positively regulate the growth of tobaacco seedlings roots under salt stress.