《植物生理学报》 2018, 54(1): 71-80

茶树胞质型谷氨酰胺合成酶基因CsGS1s的克隆及表达分析

汤丹丹^{1, 2}, 刘美雅^2,*, 张群峰², 范凯², 石元值², 马立峰², 伊晓云², 倪康², 阮建云^2,*

¹中国农业科学院研究生院, 北京100081; ²中国农业科学院茶叶研究所, 农业部茶树生物学与资源利用重点实验室, 杭州310008

通信作者：刘美雅；E-mail: 阮建云(jruan@mail.tricaas.com)、刘美雅(liumeiya@tricaas.com)

摘　要：

谷氨酰胺合成酶(glutamine synthetase, GS)是植物氮素同化过程中的限速酶。本试验选取茶树(Camellia sinensis) ‘龙井43’为材料, 利用RACE法克隆得到3个茶树CsGS1s基因成员。序列分析显示CsGS1.1、CsGS1.2和CsGS1.3基因全长分别为1 634、1 482和1 398 bp, 其开放阅读框长度均为1 071 bp, 编码356个氨基酸; CsGS1s蛋白均为亲水性、非分泌蛋白, 定位在细胞质中且无跨膜结构。序列比对显示CsGS1.1s具有GS1基因家族的特征区域。系统进化树分析表明, CsGS1.1s与拟南芥(Arabidopsis thaliana)、油菜(Brassica napus)、大麦(Hordeum vulgare)、水稻(Oryza sativa)以及玉米(Zea mays)的GS1表现出较近的亲缘关系。CsGS1s组织特异性表明, CsGS1.1和CsGS1.3在根部表达量最高, CsGS1.2主要在叶中表达, 但总体来看CsGS1.2在上述器官的表达丰度极低。利用实时荧光定量PCR对茶树在不同氮源处理下的表达进行检测, 结果表明, 在茶树叶中, CsGS1.1的表达量主要在NO₃^-处理后期才显著提高, 而CsGS1.2和CsGS1.3的表达量则主要受NH₄⁺处理的影响; 在茶树根中, CsGS1s的表达水平受铵态氮的诱导显著高于硝态氮, 尤其是CsGS1.1和CsGS1.2。

关键词：茶树; CsGS1s; NH₄⁺; NO₃^-; 表达模式

收稿：2017-06-27   修定：2017-12-04

资助：国家自然科学基金(31700614)、浙江省自然科学基金(LQ15C150004)、国家重点研发计划项目(2016YFD-0200901)和中国农业科学院科技创新工程(CAAS-ASTIP-2017-TRICAAS)。

Isolation and expression profiles of cytosolic glutamine synthetase genes CsGS1s in tea plant (Camellia sinensis)

TANG Dan-Dan^1,2, LIU Mei-Ya^2,*, ZHANG Qun-Feng², FAN Kai², SHI Yuan-Zhi², MA Li-Feng², YI Xiao-Yun², NI Kang², RUAN Jian-Yun^2,*

¹Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China; ²Key Laboratory of Tea Plant Biology and Resources Utilization (Ministry of Agriculture), Tea Research Institute, Chinese; Academy of Agricultural Sciences, Hangzhou 310008, China

Corresponding author: LIU Mei-Ya; E-mail: 阮建云(jruan@mail.tricaas.com)、刘美雅(liumeiya@tricaas.com)

Abstract:

Glutamine synthetase (GS) is the key enzyme which determines the process of nitrogen assimilation in plants. In this study, three cytosolic glutamine synthetase encoding genes (GS1) were isolated from Camellia sinensis cv. Longjing43 using RACE. The full cDNA length of CsGS1.1, CsGS1.2 and CsGS1.3 were 1 634, 1 482 and 1 398 bp, respectively and each had a 1 071 bp length open reading frame encoding a 356-amino acid protein. All these three genes CsGS1s encoded hydrophilic and non-secreted proteins which localized in the cytoplasm without transmembrane structure. Sequence alignment showed that the CsGS1.1s contained characteristic feature domains of GS1s family. The phylogenetic relationship indicated that CsGS1s and GS1s of Arabidopsis thaliana, Brassica napus, Hordeum vulgare, Oryza sativa and Zea mays were grouped into one clade. Analysis of CsGS1s tissue specificity in tea plant showed that CsGS1s were expressed in root, stem and leaf, interestingly, the transcription level of CsGS1.2 was rather low. The expression level of CsGS1.1/CsGS1.3 was highest in root while CsGS1.2 was mainly expressed in the leaf. Furthermore, the expression patterns of CsGS1s in response to different nitrogen forms in tea plant were detected using quantitative real-time PCR analysis. In leaves, the transcription of CsGS1.1 was just induced by nitrate until the 48 h treatment time point during the whole nitrogen treatment time course. Contrary, the expression of CsGS1.2/CsGS1.3 was highly induced by ammonium and with a slight induction by nitrate. In roots, the response of CsGS1s to ammonium was rather higher than that of nitrate, especially for CsGS1.1/CsGS1.2.

Key words: tea plant (Camellia sinensis); CsGS1s; ammonium; nitrate; expression pattern