向日葵V-ATPasea3亚基基因的克隆及表达分析

Cloning and Expression Analysis of V-type Proton ATPase Subunit a3 Gene in Sunflower(Helianthus annuus L.)

采用RACE技术,从向日葵P50中克隆V-ATPase a3亚基基因c DNA全长,并进行生物信息学分析;利用实时荧光定量PCR分析不同浓度、不同时间的Na Cl、ABA和PEG模拟干旱胁迫条件下V-ATPase a3亚基基因的表达特征,以及相同胁迫条件下该基因在向日葵不同器官的表达特征。序列分析表明,该基因c DNA全长2 873bp,含5'-UTR 109bp、3'-UTR 295bp及编码区2 469bp,编码822个氨基酸,其编码蛋白质的理论分子质量为204.55k Da,等电点为6.29,Gen Bank登录号为KU315054。该基因编码的蛋白质为疏水性的跨膜蛋白,亚细胞定位预测其在质膜上。向日葵V-ATPase a3亚基与已报道的10种植物的V-ATPase a3亚基的同源蛋白有高度相似的保守区域,在进化上与朝鲜蓟的亲缘关系最近。实时荧光定量PCR结果表明,向日葵受到Na Cl、ABA和PEG模拟干旱三种非生物胁迫后,V-ATPase a3亚基基因均上调表达,但表达模式不同,不同器官存在特异性表达差异。研究认为,V-ATPase a3亚基基因响应了向日葵非生物胁迫的应答,为加强对V-ATPase基因的利用奠定基础。

The full length cDNA sequence of V-type proton ATPase subunit a3 gene was cloned from sunflower P50 by RACE. The sequencing result showed that full length eDNA （2 873bp） contain the 2 469bp open reading frame （ORF）, encoding a 822 amino acids, 109bp 5＇-untranslated region and 295bp 3＇- untranslated region. It was a hydrophobic transmembrane protein and the predicted protein＇ s molecular weight was 204.55kDa, isoelectric point was 6.29, GenBank accession number was KU315054. The protein had highly similar conserved region between sunflower and other 10 kinds of plants. The genetic relationship of sunflower was closest to Cynara cardunculus var. scolymus. RT-qPCR showed that the gene was up-regulated under different concentrations of NaCl, ABA and PEG in sunflower with different expression patterns in different conditions and organs. The research suggested that the V-type proton ATPase subunit a3 gene of sunflower resoonded to abiotic stresses, which laid the foundation for strengthening its utilization.