Abstract:

V-ATPase is commonly found in plants and plays a vital role in abiotic stress tolerance. Cloning and expression analysis of the gene(s) encoding key V-ATPase subunit(s) are helpful to elucidate its regulation and response under diverse stresses. The aim of this study was to investigate expression and characterization of a V-ATPase subunit gene from Zea mays. On the basis of a SSH library constructed from maize inbred line “CN165” under drought stress, we got a uni-EST which has high similarity with other plant vacuolar H-+ATPase subunit A (VHA-A) through BLAST(NCBI) similarity analysis, thereafter the RACE technique was used to amplify the 5′ and 3′ends of H-+ATPase gene, respectively. Finally, a full-length cDNA (2 414 bp) was obtained, which contained an open reading frame (ORF) of 1 863 bp encoding a 620 aa protein, a 5′-untranslated region of 293 bp and 3′-untranslated region of 257 bp, then named ZmVHA-A. Bioinformatics methods were performed for the gene structure and mo-lecular similarity analysis. Two conserved domains, GAFGCGKT and PSVNWLISYS, interacted with the phosphate groups of the coding region, indicating the highly conserved vacuolar H-+ATPase subunit A. Homologous analysis showed that the amino acids sequence had the identity of 92.7%, 89.8%, and 89.8% with the rice, wheat, and barley H-+ATPase subunit A, respectively. To evaluate the response of ZmVHA-A gene to different abiotic stresses, transcript levels of ZmVHA-A was examined in maize. Northern blotting analysis revealed that ZmVHA-A gene was induced by water stress and showed different responses to salt, cold, and ABA treatment in maize seedling stage. The results showed that a full length H-+ATPase subunit A gene (ZmVHA-A) was gained in maize by RACE, which was a highly conservative functional subunit and participated in adaptation of V-ATPase to stress by transcriptional regulation. The ZmVHA-A gene was up-regulated under diverse abiotic conditions.

Key words:

RACE, ZmVHA-A, Abiotic stress, Expression, Zea mays L.