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

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

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Ii ESP基因的c DNA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Ii ESP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:Ii ESP基因含有2个外显子和1个内含子,其基因组、c DNA和ORF序列长度分别为1 555、1 315和1 164 bp。Ii ESP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Ii ESP与欧洲菘蓝It ESP、拟南芥At ESP和甘蓝Bo ESP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Ii ESP基因,证实Ii ESP蛋白定位于叶绿体中。RT-PCR分析结果显示,Ii ESP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。

In order to investigate the biological functions of epithiospecifier proteins（ESPs） in specifying the enzymolysis products of glucosinolate in cruciferous plants, the c DNA and the genomic DNA sequences of Ii ESP were cloned from Isatis indigotica at first, and the expression patterns of this gene were characterized subsequently. Then the subcellular localization of Ii ESP was determined by using the GFP tag and the threedimensional structure of Ii ESP was predicted based on the amino acid sequences. The results show that Ii ESP contains two exons and one intron, and its genomic DNA, c DNA and open reading frame（ORF） are 1.555, 1.315 and 1.164 bp in length, respectively. Ii ESP 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 Ii ESP with It ESP of Isatis tinctoria, At ESP of Arabidopsis thaliana and BoESP of Brassica olerace are 79%, 76% and 77%, respectively. By agroinfiltration method, Ii ESP was confirmed to be located in chloroplasts after transient expression in leaf epidermal cells of Nicotiana benthamiana. RT-PCR analyses showed that Ii ESP 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.