Clone, Expression Characterization And Over-Expressing Transgenic Study Of Tobacco NtCIOK23Gene

CBLs（calcineurin B-like proteins）belong to small protein families which areactivated by Ca2+in plant, and the Ca2+signals are transferred to the downstreamfollowing the interaction between the activated CBLs and CIPKs （CBL-interactingprotein kinase）. It was indicated that CIPKs were involved in plant growth and negativeenvironment responses, and the expression of CIPKs were affected by abiotic stressesincluding drought, salty and low-K+. It was indicated that AtCIPK23protein playedimportant role in plant potassium metabolism. The researches of CIPKs genes weremainly focused on the Arabidopsis and Rice. In other plants such as Poplar, Pea andLucerne, the exploration of CIPKs became more and more important after thecompletion of genome sequencing. Tobacco is an important model plant and economicagricultural crop, the research of tobacco CIPKs genes will have profound influencesboth on basic theory research and molecular breeding. In this study, we cloned atobacco CIPK gene named NtCIPK23via bioinformatics analysis and moleculartechniques, and we also studied NtCIPK23gene structure, expression profiles undervarious stresses, and gene function. The main work included the followings:①The ORF sequence of NtCIPK23which is1350bp in length was cloned with thehelp of bioinformatics methods. The GC-content of NtCIPK23is42.13%, the similarityof CIPK gene sequences from poplar and ricinus was as high as80%. It was predictedthat NtCIPK23encoded a protein containing450amino acid residues, which was50.702kD in molecular weight,9.18in PI, and-0.3751in the total average value ofhydrophilicity. Comparative genomics analyses in Arabidopsis, rice and other plantsrevealed that NtCIPK23was conserved both in N-terminal protein kinase domain andC-terminal NAF domain.②Semi-Quantitative RT-PCR results showed that NtCIPK23was induced bydrought, salty and SA stress. However, the expression profiles of NtCIPK23werediverse due to different stresses. NtCIPK23didn’t show the noticeable inducingexpression trends under the cold stress. These results established a fact that NtCIPK23might play a crucial role in some specific abiotic stresses.③In this study, we constructed CaMV35S promoter induced NtCIPK23over-expressing vector using plant TA cloning vector pCXSN. The constructed vectorpCXSN-35S:NtCIPK23was introduced into Agrobacterium tumefaciens LBA4404via electroporation. Through PCR and sequencing analysis methods, we obtained positiveclones. The core element of pCXSN-35S:NtCIPK23was inserted into tobacco genomeadopting leaf disc transformation method. We employed Semi-Quantitative RT-PCRand TAIL-PCR to screen the transgenic tobacco. This study laid a foundation for thefuture NtCIPK23research.