Gene Expression And Functional Analysis Of TaCIPK2 In Transgenic Tobacco

Plants are frequently challenged by abiotic stresses such as drought, high salinity, extreme temperature and so on, which bring greatly negative effects to their growth, development and reduce the yield and quality of crops. During the evolution process, plants have developed elaborate metabolisms to respond to various abiotic stresses. The CBL-CIPK complex, which composed of CBLs(calcineurin B-like protein) and its specifical interacting protein kinases CIPK, has been reported to play important roles in response to abiotic stresses. However, the functional exploration of CBL-CIPK in important crop wheat(Triticum aestivum L.) is less reported. In this study, the expression and biological functional analysis of Ta CIPK2 were investigated. The main results are as follows:(1)The expression profiles analyses of Ta CIPK2 in wheat organs and after the treatments with Na Cl, PEG, H2O2 and ABA were performed by quantitative q RT-PCR method. The results showed that Ta CIPK2 was expressed at different levels in organs examined with highest expression in leaves and stems. Ta CIPK2 expressions in response to different stresses and signaling molecules were different. All treatments of PEG, ABA, H2O2 upregulated Ta CIPK2 expressions in leaves, while the expression was not changed in root by all these treatments.(2) Subcellular localization analysis of Ta CIPK2-GFP in wheat leaf epidermal cells transformed with p BI121-Ta CIPK2-GFP via Agrobacterium-mediated transformation showed that Ta CIPK2 was localized throughout the cell.(3) Yeast Two-Hybrid Assays was performed to research the interaction between Ta CIPK2 and Ta CBLs by co-transforming the yeast expression vector p GADT7-Ta CIPK2 and p GBDT7-Ta CBLs into AH109 yeast strain, the result showed that Ta CIPK2 could interact with Ta CBL1, Ta CBL2, Ta CBL3 and Ta CBL4.(4) The recombinant plasmid p BI121-Ta CIPK2-GFP was transformed into tobacco plants(Nicotianatabacum L.cv Samsun) by Agrobacterium-mediated leaf disc transformation. Transgenic lines overexpressing Ta CIPK2 showed enhanced drought tolerance. Results of physiological indice analyses indicated that Ta CIPK2-overexpressing transgenic lines had lower ion leakage, MDA and H2O2 contents and higher antioxidant enzyme activities except peroxidase. Moreover, exogenous abscisic acid treatment resulted in lower seed germination rate and stomatal aperture in Ta CIPK2-overexpression lines, illustrating that Ta CIPK2-overexpression transgenic plants were more sensitive to exogenous abscisic acid. Osmotic stress also resulted in faster stomatal closure in Ta CIPK2-overexpresssion lines.In summary, our results indicated that Ta CIPK2 functions as a positive regulator in response to drought stress.