《植物生理学报》 2016, 52(1): 73-84

菘蓝环硫指定蛋白基因IiESP的克隆及其编码产物的亚细胞定位

马燕勤, 李典珍, 姚静雯, 李琦, 徐子勤^*

西北大学生命科学学院, 陕西省生物技术重点实验室, 西部资源生物与现代生物技术教育部重点实验室, 西安710069

通信作者：徐子勤；E-mail: ziqinxu@nwu.edu.cn

摘　要：

为了探究十字花科植物环硫指定蛋白(ESP)对硫代葡萄糖苷分解的导向作用所具有的生物学功能, 从菘蓝中克隆了IiESP基因的cDNA和基因组序列, 并分析了其表达模式。在此基础上利用GFP标签对IiESP蛋白进行了亚细胞定位, 并根据氨基酸序列预测了该蛋白的三维结构。结果表明: IiESP基因含有2个外显子和1个内含子, 其基因组、cDNA和ORF序列长度分别为1 555、1 315和1 164 bp。IiESP蛋白由387个氨基酸构成, 含有5个Kelch模块, 可折叠形成球状超螺线管结构。多重比对发现IiESP与欧洲菘蓝ItESP、拟南芥AtESP和甘蓝BoESP在氨基酸序列上的一致性分别为79%、76%和77%。通过农杆菌渗入法在本氏烟草叶片表皮细胞中瞬时表达IiESP基因, 证实IiESP蛋白定位于叶绿体中。RT-PCR分析结果显示, IiESP主要在下胚轴、茎、早期花序、成熟花序、花、萼片和花瓣中表达, 其在花被中的高活性可能与吸引昆虫进行传粉有关。

关键词：菘蓝; 环硫指定蛋白; 表达分析; 亚细胞定位; Kelch模块

收稿：2015-10-15   修定：2015-12-14

资助：国家自然科学基金(30870194和J1210063)、陕西省重点实验室科研计划(15JS111、12JS103和2010JS090)和西北大学研究生创新计划(YZZ14076和YZZ13068)。

Cloning of the epithiospecifier protein gene IiESP from Isatis indigotica and subcellular localization of the encoding product

MA Yan-Qin, LI Dian-Zhen, YAO Jing-Wen, LI Qi, XU Zi-Qin^*

Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education; Shaanxi Provincial Key Laboratory of Biotechnology; College of Life Sciences, Northwest University, Xi’an 710069, China

Corresponding author: XU Zi-Qin; E-mail: ziqinxu@nwu.edu.cn

Abstract:

In order to investigate the biological functions of epithiospecifier proteins (ESPs) in specifying the enzymolysis products of glucosinolate in cruciferous plants, the cDNA and the genomic DNA sequences of IiESP were cloned from Isatis indigotica at first, and the expression patterns of this gene were characterized subsequently. Then the subcellular localization of IiESP was determined by using the GFP tag and the threedimensional structure of IiESP was predicted based on the amino acid sequences. The results show that IiESP contains two exons and one intron, and its genomic DNA, cDNA and open reading frame (ORF) are 1.555, 1.315 and 1.164 bp in length, respectively. IiESP is constituted by 387 amino acid residues and contains 5 Kelch motifs which can fold into a spherical super-solenoid structure. Multiple alignments of the amino acid sequences indicate that the identities of IiESP with ItESP of Isatis tinctoria, AtESP of Arabidopsis thaliana and BoESP of Brassica olerace are 79%, 76% and 77%, respectively. By agroinfiltration method, IiESP was confirmed to be located in chloroplasts after transient expression in leaf epidermal cells of Nicotiana benthamiana. RT-PCR analyses showed that IiESP was mainly expressed in hypocotyl, stem, early inflorescence, mature inflorescence, flower, sepal and petal, and its higher activity in perianth was probably associated with insect attraction and entomophilous pollination.

Key words: Isatis indigotica; epithiospecifier protein; expression analysis; subcellular localization; Kelch motif