茶树硝酸盐转运蛋白基因的克隆和表达分析

汪进, 添先凤, 江昌俊, 李叶云*
安徽农业大学茶叶生物化学与生物技术教育部重点实验室, 合肥230036

通信作者:李叶云;E-mail: lyy@ahau.edu.cn;Tel: 0551-65786401

摘 要:

硝酸盐转运蛋白(NRT)是植物吸收和利用硝态氮的一种关键蛋白。运用RACE技术从茶树中扩增出NRT基因的cDNA, 并利用实时荧光定量PCR检测了CsNRT基因在不同茶树器官与品种之间的差异表达。结果表明: CsNRT基因的cDNA全长2 061 bp, 开放阅读框为1 818 bp, 编码含由605个氨基酸组成的蛋白质, GenBank登录号为KJ160503, 属于NRT2基因家族。CsNRT为组成型基因, 对不同处理的水培茶苗进行定量表达分析显示, 该基因在根、茎、叶中都有表达, 其中在根部的表达水平最高, 1.0 mmol•L-1的NO3 -可诱导其表达量上升7.53倍。不同茶树品种中CsNRT基因的表达也有较大差异, ‘龙井长叶’和‘凫早2号’的表达量较高, 前者强烈响应0.5和1.0 mmol•L-1 NO3 -的诱导, 后者的响应浓度为1.0和2.0 mmol•L-1, 而‘舒茶早’在各浓度下的表达差异不明显。

关键词:茶树; 硝酸盐转运蛋白; 基因克隆; 表达分析

收稿:2014-02-11   修定:2014-06-23

资助:国家星火计划项目(2012GA710001)和农业部“948”项目(2012-S19)。

Cloning and Expression Analysis of Nitrate Transporter Gene in Camellia sinensis

WANG Jin, TIAN Xian-Feng, JIANG Chang-Jun, LI Ye-Yun*
Key Laboratory of Tea Biochemistry and Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei 230036, China

Corresponding author: LI Ye-Yun; E-mail: lyy@ahau.edu.cn; Tel: 0551-65786401

Abstract:

Nitrate transporter (NRT) is one of the key proteins involved in nitrate nitrogen assimilation in plants. In this study, the cDNA of NRT gene in tea (Camellia sinensis) was amplified via rapid amplification of cDNA ends (RACE) technique, and their expression levels in different organs and cultivars were quantified with qPCR. The results showed that the full-length cDNA of CsNRT, containing a 1 818-bp open reading frame (ORF), was 2 061 bp encoding a polypeptide of 605 amino acids. This gene belonged to NRT2 gene family, and was named as CsNRT with the accession number KJ160503 in GenBank. CsNRT as a constitutive gene, had the high expression level in roots compared to those in stems and leaves in tea plants. In the water-cultured tea plantlets treated with medium concentration of nitrate nitrogen at 1.0 mmol•L-1, the expression level of CsNRT increased 7.53 folds than that of untreated group. Moreover, their expression levels fluctuated in different tea cultivars in which C. sinensis cv. ‘Longjinchangye’ and cv. ‘Fuzao No.2’ were higher than cv. ‘Shuchazao’. CsNTR in cv. ‘Longjinchangye’ was greatly induced by the treatment of low and medium concentrations of nitrate nitrogen at 0.5 and 1.0 mmol•L-1 respectively, while CsNTR in cv. ‘Fuzao No.2’ responded to medium and high concentrations of nitrate nitrogen at 1.0 and 2.0 mmol•L-1 respectively. However, the CsNRT expression level in cv. ‘Shuchazao’ had no obvious changes when treated with different concentrations of nitrate nitrogen.

Key words: Camellia sinensis; nitrate transporter; gene cloning; expression analysis

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