烟草NtGCN2的克隆与表达分析

张康旭, 刘国顺, 李雯雯, 王叶青, 杨永霞, 贾宏昉, 张洪映, 崔红, 张松涛*
河南农业大学烟草学院, 烟草行业烟草栽培重点实验室, 郑州450002

通信作者:张松涛;E-mail: zhangsongzi@163.com;Tel: 0371-63558121

摘 要:

GCN2是目前所有测序植物中唯一的eIF2α激酶, 它可以通过磷酸化真核翻译起始因子eIF2α来调节蛋白的合成, 从而对氨基酸的缺乏和各种胁迫做出响应。本研究采用RACE PCR技术从烟草K326中获得了GCN2的末端序列, 通过RT-PCR方法获得了部分cDNA序列, 经过拼接得到了GCN2的全长cDNA序列, 将其命名为NtGCN2 (GenBank登录号KJ706220)。分析发现, NtGCN2基因的cDNA全长为4 196 bp, ORF全长为3 759 bp, 编码1 252个氨基酸残基, 分子量为141.4 kDa, 等电点为5.58。Blastp分析结果表明, NtGCN2与土豆和番茄的同源性分别达到90%和88%。对其蛋白质结构域进行预测发现, NtGCN2包含了典型的GCN2激酶功能域。荧光定量PCR分析表明, 该基因在根、茎、叶和花中均有表达, 在叶片中表达最强, 根中的表达最弱。

关键词:烟草; GCN2; eIF2α激酶

收稿:2014-05-27   修定:2014-07-15

资助:国家自然科学基金(31100201)、河南省教育厅科学技术研究重点项目(13B210059)和河南省高等学校青年骨干教师资助计划(2013GGJs-039)。

Cloning and Expression Analysis of NtGCN2 in Nicotina tobaccum

ZHANG Kang-Xu, LIU Guo-Shun, LI Wen-Wen, WANG Ye-Qing, YANG Yong-Xia, JIA Hong-Fang, ZHANG Hong-Ying, CUI Hong, ZHANG Song-Tao*
Key Lab for Tobacco Cultivation of Tobacco Industry, College of Tobacco Science, Henan Agricultural University, Zhengzhou 450002, China

Corresponding author: ZHANG Song-Tao; E-mail: zhangsongzi@163.com; Tel: 0371-63558121

Abstract:

Higher plants contain only a GCN2-type eIF2α kinase in their genome, which can phosphorylate the translation initiation factor eIF2α to regulate the general protein synthesis in response to amino acid starvation or stresses. In this study, we cloned the GCN2 from Nicotina tobaccum K326 by rapid amplification of cDNA ends (RACE), named NtGCN2, which included a 3 759-bp open reading frame encoding 1 252 amino acid residues. The molecular weight of the predicted amino acid sequence of the NtGCN2 protein was 141.4 kDa with a theoretical pI of 5.58. Further, sequence alignment by BLASTP showed NtGCN2 shared the high similarity to Solanum tuberosum GCN2 (90%) and Solanum lycopersicum GCN2 (88%) and contained a typical kinase catalytic domain and a His-tRNA synthetase-related domain. Finally, Real-time PCR was porfermed to reveal that NtGCN2 was expressed in roots, stems, leaves and flowers, with higher expression in leaves and lower in roots.

Key words: tobacco; GCN2; eIF2α kinase

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