Study On The Mechanism Of Rice OsCBLs-oSMTN Interaction

Calcium is an important second Messenger in plant stress. A variety of calcium signals triggered by environmental stress, couled be recognized and decoded by specific plant cell calcium receptor, and through protein interactions to transduct calcium signal, therefore produces a set of physiological and biochemical reactions. Calcineurin B-like proteins(AtCBL) is a calcium-sensing proteins specific to the plant.. Since the AtCBL-AtCIPK signalling systems were found,AtCIPK has been considered as the only target protein of AtCBL and participated in calcium signaling pathway caused by series of environmental changes through protein interactions. However, recently Seung-Ick.et al, through yeast two-hybrid experiments and Bimolecular fluorescence complementation experiments confirmed that the Arabidopsis AtCBL3protein member of this family has a novel interaction partner,5’-methylthioadenosine nucleosidase AtMTN1, which is one key enzyme in Methionine-recycling pathways. And AtCBL3regulating the biosynthetic pathway of ethylene and polyamines in plant growth processes through interaction with AtMTN1.Transgenic plants(T3) overexpression sense OsMTN gene、overexpression OsCBL3gene and T-DNA insertion mutant strains of OsCBL3have been obtained from previous studies. Preliminary analysis of endogenous gene expression in transgenic rice and effect of seedling salt resistance and drought tolerance. We test the salt and drought tolerance.Semi-quantitative RT-PCR comfirmed that the expression of OsMTN and OsCBL3remarkable augmented in lots of sense of OsMTN and OsCBL3transgenic seedings,respectively,and the expression of OsCBL3in T-DNA insertion mutant strains of OsCBL3decreased significantly.To research the specific function of OsMTN and OsCBL3in response to salt and mannitol stress.we cultivation the transgenic lines in medium for7days.The results comfirmed that some of OsMTN-overexpressing plant and OsCBL3-overexpressing plant showed reduced capacity to endure salt stress, and the seedlings showed reduced capacity to endure mannitol stress to some extent. In addition, T-DNA insertion mutant strains of OsCBL3showed reduced capacity to endure salt stress, and the seedlings showed reduced capacity to endure mannitol stress to some extent, but the roots showed no diversity factor contrasted with wild type.To uncover the interaction localization of rice OsCBL、OsCBL3、OsCBL6with OsMTN, we carried out subcellular localization assay and the BiFC. We watched a cyclic annular distribution of OsMTN::GFP、OsCBL2::GFP、OsCBL3::GFP CBL6::GFP at the plasma membrane and tonoplast. The findings confirmed that OsMTN-OsCBLs decipher calcium signals at the vacuole, and at the plasma membrane. In addition, the interference assays by SarH74L and Brefeldin A comfirmed that, the target pathway of OsMTN/OsCBLs protein was neither COPI nor COPII pathway. Both bimolecular fluorescence complementation assay and the yeast two hybrid interaction analyses showed that the OsCBL3W mutant containing the91amino acids between the109th and199th amino acid residues of OsCBL3retained the ability to interact with OsMTN. This results provides a theoretical basis for further revealing the interaction mechanism of OsCBL3and OsMTN protein.