Cloning and transformation of AtCDPK to tobacco

Enviromental stresses are the most important factors limiting crops production worldwide. Being a member of CDPK family, AtCDPK5 gene plays an important role in plant defense responses to abiotic stresses. The objective of this study was to clone AtCDPK5 and transfer it to tobacco in order to increase abiotic stress resistance. Total RNA extracted from Arabidopsis thaliana leaves and converted into cDNA. AtCDPK5 was amplified by PCR with specific primers and transformed to pGEM-T easy vector and cloned in E.coli. The integrity of the construct was verified by colony PCR on the clones grown in LB medium containing Ampycilin. The gene fragment was inserted into pBI121 vector and then cloned in E.coli. The clones were grown in LB medium containing kanamycin and the integrity of the construct was verified by colony PCR. The recombinant plasmid was introduced into Agrobacterium tumefaciens. The constructions were transformed by Agroinfection method into of tobacco explants. After plant adaptation period, plants were moved into pots and kept in greenhouse. Transgenic plants were selected by Kanamycin, Sephotaxim and Rifampsin resistance. Trasformation was also verified on cDNA level by PCR with specific primers.