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园艺学报 ›› 2015, Vol. 42 ›› Issue (1): 95-103.doi: 10.16420/j.issn.0513-353x.2014-0503

• 其它园艺植物 • 上一篇    下一篇

茶树NADPH氧化酶基因的克隆、亚细胞定位与表达分析

王明乐, 王伟东, 赵真, 黎星辉   

  1. 南京农业大学茶叶科学研究所,南京 210095
  • 出版日期:2015-01-25 发布日期:2015-01-25
  • 基金资助:

    国家现代农业产业技术体系建设专项资金项目(CARS-23);国家自然科学基金项目(31470690);苏州市科技项目(SZGD201067)

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

WANG Ming-Le, WANG Wei-Dong, ZHAO Zhen, LI Xing-Hui   

  1. Tea Research Institute,Nanjing Agricultural University,Nanjing 210095,China
  • Online:2015-01-25 Published:2015-01-25

摘要: 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CsRBOHA的cDNA全长(GenBank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 kD,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 NaCl)、ABA(200 mg ? L-1)和干旱(10% PEG 6000)条件下出现不同程度的上调。

关键词: 茶, NADPH氧化酶, 基因克隆, 亚细胞定位, 实时荧光定量, 表达分析

Abstract: In this study,the homologous gene CsRBOHA was isolated from tea plant(Camellia sinensis)by RACE and RT-PCR. The full length of CsRBOHA gene(GenBank 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 kD and the pI value was 9.28. Homologous alignment showed that the predicted CsRBOHA protein was similar to Nicotiana tabacum and Ricinus communis,with the similarity of 79%. CsRBOHA 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,NaCl(200 nmol ? L-1),ABA(200 mg ? L-1)and 10% PEG 6000 at different degree.

Key words: tea plant, NADPH oxidase, gene cloning, subcellular localization, qRT-PCR, expression analysis

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