Cloning And Expression Analysis Of ZmVHA-A And ZmVHA-B Genes Related To Drought In Maize(Zea Mays.L)

The plant V-ATPase is a membrane-bound transport protein located to the tonoplast and various subcellular membranes, including the Golgi, endoplasmic reticulum, intracellular vesicles, as well as the plasma membrane. The V-ATPase uses the energy released during cleavage of theγ-phosphate group of cytosolic ATP to pump protons into the vacuolar lumen and acidify the vacuole, thereby creating an electrochemical H~+-gradient which is the driving force for a variety of transport events of ions and metabolites. So V-ATPase is indispensable for plant growth, development and adaptation to changing environmental conditions.Although genes encoding V-ATPase subunits have been detected in many plants, and we knew its structure and composition, But expression and regulation of V-ATPase only had a little report In the early stage of our research we found that expression of vacuolar H~+-ATPase subunit A gene was induced in drought stress. Based on a uni-EST from a SSH library built from maize inbred line"CN165"under drought stress, the complete cDNA sequence of VHA-A gene(vacuolar H~+-ATPase subunit A) from CN165, named ZmVHA-A, was obtained using RACE(Rapid Amplification of cDNA Ends) technique and by using RT-PCR we also got ZmVHA-B gene encoding V-ATPase B subunit. To elucidate its regulation and response to environmental stress, Northern blotting analysis of ZmVHA-A and ZmVHA-B genes was carried out under water stress, salt stress, cold stress and exogenous ABA in seedling stage and under drought stress in flowering stage. then we performed the subcellular localization research by using the fuse expression analysis of ZmVHA-A and GFP in onion epidermal cells. Finally overexpression of ZmVHA-A gene was carried out in Arabidopsis thaliana.The main findings were as follows:1. using RACE(Rapid Amplification of cDNA Ends) strategy, we got all length cDNA sequence of ZmVHA-A gene, and Sequence analysis indicated the full-long cDNA fragment is 2414bp, including the 5'-UTR(293bp), the 3'UTR(257bp) and the coding region(1863bp), ZmVHA-A was predicted to encode a protein of 620 amino acids, isoelectric point of 5.36. Meanwhile the coding sequence of ZmVHA-B gene was obtained by RT-PCR and sequence analysis showed that it encoded 487 amino acids, protein molecular weight of 54.09kD, isoelectric point of 4.99.2. Amino acid sequence analysis and cluster analysis showed V-ATPase subunit A and B are highly conserved protein subunit, and V-ATPase subunit A and B homology is highly relevant to the level of biological evolution. Homologous analysis of A subunit showed that it respectively presents 92.7%,89.8% and 89.8% identity on the ammo acids peptide with oryza,wheat and barley, there are two conserved domains which interact with the phosphate groups of ATP(GTP) in coding region of subunit A:"GAFGCGKT"and"PSVNWLISYS". B subunit also detected a conservative domain that play its essential role in functional regulation of V-ATPase.3. To examine the subcellular localization of the ZmVHA-A protein in plant cells,we fused the coding region of ZmVHA-A in frame to the coding region for the N-terminal side of green fluorescent protein(GFP), and the fusion gene was expressed under the control of the 35S promoter of Cauliflower mosaic virus(CaMV). Then fused expression of ZmVHA-A and GFP was carried out in onion epidermal cells, the results showed: When only GFP was introducde into the onion epidermal cells, after incubation at 25℃for 24-36 hours GFP fluorescence was observed in Plasmid membrane,Nuclear and cytoplasm in onion epidermal cells; while fluorescence signal of the fusion protein was detected in cytoplasm, the result indicated ZmVHA-A gene was mainly expressed in cytoplasm.4. Northern blotting results showed ZmVHA-A and ZmVHA-B genes responsed to drought, high salt, low temperature and ABA treatment. Firstly,ZmVHA-A and ZmVHA-B genes were induced by drought stress in seedling stage and its expression was not in proportion to RWC of leaf; Secondly, expression of ZmVHA-A and ZmVHA-B genes can be induced by ABA,Thirdly, under drought and high salinity of seedling, ZmVHA-B gene displayed two transcripts inducible expression, and the same event was detected in silk of drought stress; The isoform would play an important role in stress conditions, our research results indicated that V-ATPase could participate in the regulation of responses by certain common pathway in adverse conditions.