Functional Identification Of LeCBLs From Tomatoes(Lycopersicon Esculentum)

Ca2+serving as second messeage plays the key roles in responses to stresses, each of calcium signals resulting from spatiotemprol patterns and the extent of these changes in Ca2+concertration has been implicated as a specific stress. CBL, universal a kind of Ca2+sensor in plants, interacting with CIPKs by binding NAF domains of CIPKs following activing CIPKs couple with stresses.Although the progress have been made in studying CBLs from Arabidopsis and Oryza sativa only have some papers been published in Zea mays, Sorghum bicolor, Anemone vitifolia Buch, and so on. It is necessary to conduct research on CBL in every specie since not all CBLs which are high identical between species are same function. Tomato is one of the model plants to study gene function while functions of CBLs have not been well characterized. Carrying out studying of CBLs is benefit for discovering characterization of CBL genes in tomato and supporting theoretical for improving the tomato increasing stresses resistance. Results are as follows:1Five LeCBLs (LeCBL2、LeCBL3、LeCBL4、LeCBL9and LeCBL10) over-expression and four LeCBLs (LeCBL2、LeCBL3、LeCBL9and LeCBL10) RNAi vectors were constructed. Then these vectors were transformed into the model plants Nicotiana benthamiana and Lycopersicon esculentum by Agrobacterium tumefaciens GV3101. T1resistant Nicotiana benthamiana and TO resistant Lycopersicon esculentum seeds were obtained.2LeCBL8over-expression and RNAi vectors which have been constructed were transformed into Nicotiana benthamiana and Lycopersicon esculentum. T1resistant Nicotiana benthamiana and TO resistant Lycopersicon esculentum seeds were obtained. In addition, we analysed the LeCBL8resistant tobacco (the wide type is W38) under cold, salt, high pH and low pH. Resistance to high concertration of salt was confirmed and LeCBL8may also play the roles in resistance to cold, salt, high pH and low pH.3. Bimolecular fluorescence complementation (BiFC) is a novel technology to visualize protein-protein interactions To provide multiple options in the study of protein interactions that utilise BiFC, we reconstructed a new pair of BiFC vectors, pCAMBIA1301-nEYFP and pCAMBIA1301-cEYFP. Fluorescence can be imaged when AtCBLl and AtCIPK23are co-injected, but imaging can not be done when co-injecting AtCBL1and AtCIPK23-NAF-deleted (AtCIPK23m), suggesting that the proposed modified vector system is effective for the study of protein interactions. Applied the recontructed BiFC platform, we also analyze the LeCBLs and LeCIPKs interaction successfully. The interaction of LeCBL3-LeCIPK8, LeCBL9-LeCIPK8, LeCBL9-LeCIPK23, LeCBL9-LeCIPK24and LeCBL10-LeCIPK24are strongest, LeCBL3-LeCIPK23, LeCBL3-LeCIPK24, LeCBL4-LeCIPK3, LeCBL4-LeCIPK8, LeCBL4-LeCIPK23, LeCBL4-LeCIPK24, LeCBL9-LeCIPK3, LeCBL10-LeCIPK8and LeCBL10-LeCIPK23are fairly strong, while LeCBL3-LeCIPK3and LeCBL10-LeCIPK3are weakest。...