Isolation, Expression Analysis And Genetic Transformation Of The TaCIPK16 Gene In Wheat(Triticum Aestivum L.)

In recent years, CBL(Calcineurin B-like Protein) has been named as a kind of Ca2+ sensor which is peculiar to the plants. The CIPK protein(CBL-Interacting Protein Kinase) is the protein kinase interacted with the CBL. The CBL and CIPK constitute the Ca2+-CBL-CIPK signal system that plays a key role in response to the abiotic stress such as drought, high salt and ABA signaling pathway. In this study the full-length cDNA sequence of TaCIPK16 gene from the wheat has been cloned. And the main researchs are as follows:(1) Wheat ESTs of TaCIPK16 gene were searched from the databases and were assembled into full-length cDNA sequence of TaCIPK16 gene, which was amplified by using specific RT-PCR primers.(2) Based on the bioinformatics analysis of TaCIPK16 gene, TaCIPK16 was classified into the SnRK3 kinase family, and which contains N-terminal protein kinase domain and C-terminal NAF domain, PPI domains. Sequence alignment and the phylogentic analysis of the TaCIPK16 gene indicated that it had the highest homology to OsCIPK16 and was desinated as TaCIPK16.(3) Using Quantitative Real-Time PCR techneque(qRT-PCR), the expression of TaCIPK16 gene in response to the abiotic stresses including NaCl, PEG, H?O? and low temperature as well as ABA and ETH were analysised. The results showed that TaCIPK16 gene was up-regulated induced by NaCl, PEG, ETH and H?O?, indicating that TaCIPK16 gene was involved in the signal transduction of salt stress, drought stress, ethylene stress and oxidative stress as a stress-responsed gene.(4) Yeast Two-Hybrid Assays was performed to detect the interaction between TaCIPK16 and TaCBLs in yeast by constructing the pGADT7-TaCIPK16 yeast expression vector. The results showed that the TaCIPK16 protein had no interaction with the seven TaCBLs in previous study.(5) The eukaryotic expression vector of pBI121-TaCIPK16-GFP was construted for the subcellular localization analysis and the genetic transformation of tobacco.The recombinational plasmid was transient expressed in onion epidermal cells by biolistic bombardment. The results showed that the green fluoresenc protein could be observed in the cell membrane, cytoplasm and nucleus, indicating no obvious subcellular localization of TaCIPK16 protein And the agrobacterium-mediated transformation was performed to generate overexpression transgenic plants of TaCIPK16 gene in tobacco. The positive transgenic tobacco plants were obtained through the tissue culture techneque.The results are helpful for the further study of TaCIPK16 gene in response to abiotic stresses and will lay the foundation for the molecular breeding research and development of wheat.