玉米液泡ATP酶亚基A基因的克隆及表达分析

Cloning and Expression Analysis of a Vacuolar ATPase Subunit A Gene in Maize

V-ATPase在植物应对非生物胁迫中起着十分重要的作用,对其关键亚基基因的克隆及分析有助于阐释其在逆境下的调节及响应机制。本文利用RACE技术从玉米自交系CN165花期水分胁迫下的总RNA中克隆出液泡ATP酶亚基A基因,命名为ZmVHA-A。该基因cDNA全长2414bp,含5′-UTR(293bp)、3′-UTR(257bp)及编码区(1863bp),编码620个氨基酸。序列比对及结构域预测结果表明,ZmVHA-A基因编码的氨基酸序列与亲缘关系比较接近的禾本科作物如水稻、小麦和大麦的氨基酸序列同源性分别达92.7%、89.8%和89.8%,且在编码蛋白的氨基酸序列上有两个与核苷酸的磷酸基团相互作用的保守结构域"GAFGCGKT"和"PSVNWLISYS",可见该亚基是相对保守的。Northern杂交分析结果表明,ZmVHA-A基因在花丝中被水分胁迫诱导表达且在苗期对盐胁迫、冷处理和ABA处理具有不同的表达响应方式,因此认为VHA-A基因是一个比较保守的功能亚基,并且通过转录表达调节参与了V-ATPase对逆境胁迫的适应。

V-ATPase is commonly found in plants and plays a vital role in abiotic stress tolerance. Cloning and expression analy-sis 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 （2414 bp） was obtained, which contained an open reading frame （ORF） of 1863 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 molecular 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.