茶树NADPH氧化酶基因的克隆、亚细胞定位与表达分析

Molecular Cloning,Subcellular Localization and Expression Analysis of NADPH Oxidase Gene from Tea Plant

以茶树(Camellia sinensis)‘迎霜’为试验材料,采用同源克隆的方法,利用RACE和RT-PCR技术获得茶树NADPH氧化酶基因Cs RBOHA的c DNA全长(Gen Bank登录号:KJ782632)。该基因全长3 157 bp,开放阅读框2 769 bp,编码922个氨基酸。生物信息学分析显示,该基因编码的蛋白分子量为103.33 k D,理论等电点为9.28;C端序列较保守,N端序列保守性较低,与烟草和蓖麻的相似性达79%,进化关系最近;蛋白结构具有典型的家族特征。该蛋白分布于细胞质膜上,与预测结果一致。实时荧光定量PCR结果显示,该基因的表达存在组织特异性,并且在低温(4℃)、高盐(200 mmol·L-1 Na Cl)、ABA(200 mg·L-1)和干旱(10%PEG 6000)条件下出现不同程度的上调。

In this study,the homologous gene Cs RBOHA was isolated from tea plant(Camellia sinensis)by RACE and RT-PCR. The full length of Cs RBOHA gene(Gen Bank Accession No. KJ782632)was 3 157 bp,which had an open reading frame of 2 769 bp,encoding 922 amino acids. The molecular weight of the predicted enzyme was 103.33 k D and the p I value was 9.28. Homologous alignment showed that the predicted Cs RBOHA protein was similar to Nicotiana tabacum and Ricinus communis,with the similarity of 79%. Cs RBOHA was conserved with NADPH oxidase superfamily. Subcellular localization indicated that the protein was localized in plasma membrane,which is identical to the predicted results. Quantitative real-time PCR showed that the gene was tissue-specific and up-regulated by low temperature,Na Cl(200 mmol · L-1),ABA(200 mg · L-1)and 10% PEG 6000 at different degree.