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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 estrogen-targeting protein (ESP) on the guiding role of glucosinolate breakdown, the c DNA and genomic sequences of Ii ESP gene were cloned and analyzed for its expression mode. On this basis, Ii ESP protein was subcellularly localized by GFP tag and the three-dimensional structure of the protein was predicted by amino acid sequence. The results showed that the Ii ESP gene contained two exons and one intron, and the lengths of the genome, c DNA and ORF were 1 555, 1 315 and 1 164 bp, respectively. The Ii ESP protein consists of 387 amino acids and contains five Kelch modules that fold to form a spherical, super-solenoid structure. Multiple alignment revealed amino acid sequence identity of 79%, 76% and 77% for Ii ESP and European blue-collar It ESP, Arabidopsis At ESP and Chinese cabbage Bo ESP, respectively. The Ii ESP gene was transiently expressed in N. benthamiana leaf epidermal cells by Agrobacterium infiltration, confirming that the Ii ESP protein is localized in the chloroplast. RT-PCR analysis showed that Ii ESP was mainly expressed in hypocotyls, stems, early inflorescences, mature inflorescences, flowers, sepals and petals, and its high activity in perianth may be related to attracting insects for pollination.