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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 of V-ATPase a3 subunit gene was cloned from P50 sunflower by RACE technique and bioinformatics analysis was performed. Real-time fluorescence quantitative PCR was used to analyze Na Cl, ABA and PEG at different concentrations and different times to simulate drought stress conditions The expression characteristics of the V-ATPase a3 subunit gene and the expression characteristics of this gene in different organs of sunflower under the same stress were analyzed. Sequence analysis showed that the cDNA of this gene was 2 873 bp in length, containing 109bp of 5’-UTR, 295bp of 3’-UTR and 2 469bp of coding region, encoding 822 amino acids. The theoretical molecular mass of this gene was 204.55 kDa, Point 6.29 and Gen Bank accession number KU315054. The gene encodes a hydrophobic transmembrane protein, which is predicted to be localized on the plasma membrane. The sunflower V-ATPase a3 subunit has highly similar conserved regions with the homologous proteins of the V-ATPase a3 subunit of the 10 reported plants, and has the closest phylogenetic relationship to artichoke. Real-time quantitative PCR results showed that the expression of V-ATPase a3 subunit gene was up-regulated by three kinds of abiotic stresses of NaCl, ABA and PEG simulated drought in sunflower, but the expression patterns were different. Studies suggest that the V-ATPase a3 subunit gene responds to the sunflower abiotic stress response, to lay the foundation for the use of V-ATPase gene.