Cloning And Functional Analysis Of Gene VOZ1 In Rice

Ca2+, a very important second messenger, acts as a mediator of stimulus-response coupling diverse cellular function, which regulates a variety of physiological and biochemical response of cells. Response of plants to various stimuli can be detected by measuring changes of Ca2+ peak in plant cells. Calmodulin (CaM) is a small acidic protein which presents in all eukaryotes. Calmodulin (CaM), as a universal and highly conserved calcium receptor protein, is an EF-hand protein. CaM itself has no activity, but it can interact with CaMBP through calmodulin binding domain (CaMBD). The generalized calmodulin binding protein (CaMBP) is all the proteins that can bind to CaM.The CaMBP including various enzymes which activity is direct regulated by CaM and the protein which can bind to CaM in vivo or in vitro. The CaMBP is widely present in plant and animal cells, and play an important role in plant growth and stress response.NAC is a large member of the transcription factors, which include the NAM, ATAF, CUC and other subfamily. It is not only associated with the development of plants, but also connected with biotic and abiotic stress. VOZ (Vascular plant One-Zinc finger) is a zinc finger protein in vascular plants. VOZ is a member of NAM superfamily in NAC family. In this study,we take yeast two-hybrid experiment using OsCaMBP as a bait to screen the proteins interacting with OsCaMBP in chilling and drought cDNA library in rice.This study selected one protein OsVOZ1, study and explore its features in rice, in order to provide the basis for further improving of rice Ca2+-mediated stress signaling networks. The main findings are as follows:1. Analyzing the sequence of the OsVOZ1, we found that the open reading frame(ORF) of the gene is 1290 bp, encodes 429 amino acid residues, and isoelectric point is about 4.88, molecular weight is predicted to be 48.11 KD. Predicted the function of the protein found that 148 amino acids sequence have a zinc finger, the 211-304 have a NAM domain. Thus, the gene OsVOZ1 belongs to the NAC family.2. We analyzed OsVOZ1 expression levels at different tissues under different stresses by RT-PCR. We found that OsVOZ1 express in different degrees in roots, stems and leaves, and that the gene is induced by a variety of stresses, indicating that the gene belongs to a multiple stress-inducible gene (Abrrerived:Multiple stress-inducible genes, MSI).3. We got the homozygous overexpression and knock-down transgneic lines; stress treatment experiments indicate that overexpression lines enhanced resistance to salt stress.4. Subcellular localization confirmed OsVOZ1 gene is located in the cytoplasm, and the related report showed it plays a role in the nucleus, suggesting that it may be biding the outside shuttle protein of nuclear and then transferred to the nucleus play a role through a certain channel.5. We successfully expressed and purificated the fusion proteins of OsVOZl through E. coli expression system, for further verify the interaction between OsVOZl and OsCaMBP.