西兰花(Brassica oleracea var.italica)硫代葡萄糖苷酶基因cDNA分子克隆及其特征分析

Molecular Cloning and Characteristic Analysis of the cDNA Encoding Myrosinase from Broccoli

目的:研究西兰花硫代葡萄糖苷酶(简称硫苷酶)cDNA的结构特征,为硫苷酶异源表达打下基础。方法:以西兰花幼苗为试材,根据同源序列法PCR扩增硫苷酶特异cDNA片段,再用3′RACE和5′RACE分别扩增cDNA3′端序列和5′端序列,经序列拼接获得西兰花硫苷酶全长cDNA序列。利用在线蛋白质分析系统对其编码的氨基酸序列进行结构特征分析,在NCBICDD数据库中查找保守结构域,在SWISS-MODEL上进行在线蛋白同源模建,用MEGA3.1构建NJ系统树。结果:该基因cDNA全长1830bp,含有一个1641bp的ORF及31bp的5′端非翻译区和158bp的3′端非翻译区,命名为BoMyr2(GenBank登录号为EU004075);编码氨基酸序列含有20个氨基酸长度的信号肽及9个天冬酰胺N末端糖基化位点,编码的蛋白分子质量(Mw)约为62.2kD,等电点(pI)为8.71;BoMyr2推测氨基酸序列含有糖基水解酶家族1特有的糖基水解酶结构域,其编码蛋白与PDB库中白芥硫苷酶有类似的三维结构,具有明显的β/α(TIM)桶结构;BoMyr2应归为硫苷酶基因家族的MB亚族。

Objective： Molecular clone and analyze sequence characteristics of the cDNA encoding myrosinase from Broccoli （Brassica oleracea var. italica）, for the sake of study it＇s expression in yeast and make further improvement on the phenomenon that myrosinase in Brassicaceae is encoded by a gene family and it＇s expression and distribution varied in a temporal and tissue-specific manner. Methods： Using 3＇RACE and the corresponding 5＇RACE technique obtained the full-length cDNA of Broccoli homologous gene of Brassicaceae myrosinase （BoMyr2）. Nucleotide and deduced amino acid sequence of the myrosinase cDNA were analyzed on the network servers. NCBI CDD database was used for motif scan. Neighbor-joining tree was constructed by MEGA 3.1 using the amino acid sequence of myrosinase from different species of Cruciferae. The 3 D structure of broccoli myrosinase was predicted by SWISS-MODEL Conclusion： The 1 830 bp cDNA contained an ORF of 1 641 bp, which coded for a polypeptide of 548 amino acids. The deduced polypeptide had a predicted molecular weight of 62.2 kD, a pI of 8.71. The predicted amino acid sequence include a signal peptide, a characteristic domain of the gtycosyl hydrolase family 1 which include Glycosyl hydrolase family 1 N-terminal signature （FiFGvAsSAYQiEgG） and Glycosyl hydrolase family 1 active site （IYITENGFS）, nine N-glycosylation sites and revealed closest homology with myrosinase from MB subfamily. Homology model of the myrosinase from Broccoli was obtained with MOE on the base of the crystal structure of myrosinase from S. alba as template. Putative three-dimensional structures of the myrosinase showed a （β/α）8 barrel structure common to other glucosidases.