萼脊兰SjHMGR基因克隆及表达分析

Cloning and Bioinformatics Analysis of SjHMGR Gene in Sedirea Japonica

为探究兰科植物的花香基因,拓展兰科植物在花香分子育种方面思路。以萼脊兰花瓣为实验材料,按照NCBI上登录的兰科植物的HMGR基因序列设计引物,采用RT-PCR和RACE技术成功克隆萼脊兰3-羟基-3-甲基戊二酰辅酶A还原酶基因(SjHMGR)。采用生物信息学方法,利用内参基因EF1a,对SjHMGR基因的时空表达特性进行分析研究。结果显示,获得的SjHMGR基因全长为1892 bp,开放阅读框为1689 bp,编码367个氨基酸,登录号为MK448292;SjHMGR有3个HMG-COA特殊位点,且属于HMG-COA超基因家族;SjHMGR编码的蛋白为亲水性蛋白;亚细胞定位于线粒体;蛋白质2级结构分析发现,SjHMGR蛋白具有α-螺旋、延伸链和不规则折叠。SjHMGR编码蛋白质的功能预测发现,SjHMGR在中间代谢起到非常重要的角色;同源性分析与系统进化树分析发现,萼脊兰SjHMGR蛋白与兰科的进化距离最近,在同1个分支上。qRT-PCR结果显示,SjHMGR基因在根和叶中的表达量很低,在萼片和花瓣中的表达较高,具有时空特异性。

In order to explore the floral fragrance genes and develop ideas for molecular breeding of floral fragrance of orchid plants, the petals of Sedirea japonica were used as the experimental materials, and primers were designed according to the HMGR gene sequence of orchid plants registered in NCBI. The 3-hydroxy-3-methyl-glutaric coenzyme A reductase gene(SjHMGR) was cloned successfully by RT-PCR and RACE. The bioinformatics of SjHMGR gene was analyzed. The spatiotemporal expression characteristics of SjHMGR gene were analyzed using the internal reference gene EF1 a. The results showed that the length of SjHMGR gene was 1892 bp, the open reading frame was 1689 bp, encoding 367 amino acids, and the login number was MK448292. SjHMGR had three special loci of HMG-COA and belonged to the HMG-COA supergene family. The SjHMGR was a hydrophilic protein in mitochondria. Alpha helix elongated chain and irregular folding were found in the secondary structure. Functional prediction of SjHMGR-encoded proteins revealed that SjHMGR played an important role in intermediate metabolism. Homology analysis and phylogenetic tree analysis showed that the homology of SjHMGR protein was the closest to the evolution of Orchidaceae and on the same branch. The results of qRT-PCR showed that the expression of SjHMGR gene in roots and leaves were very low, and that in sepals and petals was high, which had spatial and temporal specificity.