Cloning Of Some ZmCBL Genes From Maize (Zea Mays. L) And Their Transfering Into Arabidopsis

Drought, low temperature, high salinity and other abiotic stresses severely inhibit plant growth and development.Recently, It has been demonstrated that calcineurin B-like proteins(CBLs) play important regulatory roles in the response of plants to multiple abiotic stresses. There exists at least nine CBL genes in maize genome. In the present study, the complete coding region cDNAs of three maize CBL genes, namely ZmCBL2-2, ZmCBL6 and ZmCBL8, were cloned and constructed to plant expression vector and transfered into Arabidopsis thaliana. The work laid a foundation for the analyses of the ZmCBLs'function in the future, and the main results are presented as follows.1. The complete coding region cDNAs of ZmCBL2-2, ZmCBL6 and ZmCBL8 were amplified by reverse transcription polymerase chain reaction (RT-PCR ), and each of them consists of 678, 672 and 645 base pairs and encodes a polypeptide composed of 225, 223 and 214 amino acids, respectively.2. Based on bioinformatics analyses, all of the ZmCBL2-2, ZmCBL6 and ZmCBL8 proteins contain four EF-hand motifs, and the ZmCBL8 protein also has a N-terminal myristoylation sequence.The proteins share 93%,93% and 78% sequence identity with the rice OsCBL2, OsCBL6 and OsCBL8 proteins rspectively. All the proteins were predicted to be suloble proteins with strong hydrophilicity and most probably located in cytoplasm.3. The complete coding region cDNA of ZmCBL2-2, ZmCBL6 and ZmCBL8 was inserted into the plant expression vector pCAMBIA3301 respectively and promoted by 35S promoter, and then was transferred into the competent cells of Agrobacterium tumefaciens strain GV3101 by freeze-thaw method.4. The genetic transformation of wild-type Arabidopsis thaliana was conducted with Agrobacterium strain GV3101 harboring the vector pCAMBIA3301 that carried cDNA of ZmCBL2-2, ZmCBL6 or ZmCBL8, respectively. After screening with phosphinothricin(PPT) and confirmation by polymerase chain reaction (PCR), T1 and T2 seeds were obtained from ZmCBL2-2- transformed plants. For ZmCBL6 and ZmCBL8, only T1 seeds were collected from the transformed plants.