‘西伯利亚’百合LiMCT基因的克隆及表达特性分析

Cloning and Expression Characteristic Analysis of LiMCT Gene in Lilium'Siberia'

2-C-甲基-D-赤藓醇-4-磷酸胱氨酰转移酶(2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase,MCT)是单萜合成途径中的关键酶。为揭示其在‘西伯利亚’百合(Lilium'Siberia')中的作用,明确其表达模式,本试验克隆了‘西伯利亚’百合LiMCT基因,进行同源氨基酸序列的多重比对,生物信息学分析及亚细胞定位。最后采用荧光定量PCR技术(RT-qPCR)对该基因进行时空表达分析。结果表明,LiMCT基因开放阅读框为897 bp,编码298个氨基酸,与日本樱花、蔓花生、小兰屿蝴蝶兰、莴苣、铁皮石斛的MCT蛋白相似度较高。LiMCT蛋白定位在拟南芥原生质体的细胞膜中。在不同花期中,LiMCT基因表达量在半开期和盛开期最高,花蕾期最低。在不同花器官组织中,LiMCT基因在花柱中表达最高,外花被片和花药中较多,在其他部位表达较低。本研究探究了LiMCT蛋白的结构功能及其基因在开花期的时空表达模式,为其在‘西伯利亚’百合单萜合成及其花香释放的调控机制提供科学依据。

2-C-methyl-D-erythritol-4-p hosphate cytidylyltransferase(MCT)is a key enzyme in monoterpene synthesis pathway.In order to explore its role and expression patterns in Lilium’Siberia’,LiMCT gene have been cloned,then multiple comparisons of homologous amino acid sequence and the bioinformatics analysis were carried out.We also made subcellular localization of the protein LiMCT.The temporal and spatial expression pattern was analyzed by RT-qPCR technique.The results showed that the open reading frame of LiMCT was 897 bp,which encoded 298 amino acids.There was relatively high similarity between LiMCT protein and MCT protein of Prunus yedoensis,Arachis duranensis,Phalaenopsis equestris,Lactuca sativa,and Dendrobium catenatum.The LiMCT protein was localized in the cell membrane of the protoplasts of Arabidopsis thaliana.Among the different flowering stages,the highest expression level of LiMCT appeared at early flowering and flowering periods,and at the bud period the expression level was the lowest.Among different organs and tissues,the highest expression level occurred in the style followed by outer petal and anther,and low expression levels were found in other.This study clarified the structural function of LiMCT protein and the expression patterns of the gene during flowering,and provided a basis for its regulation mechanism of monoterpene synthesis pathway and floral release in Lilium’Siberia’.