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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: To study the structural characteristics of broccoli glucosidase (thiolase) cDNA and lay a foundation for the heterologous expression of thioamidase. Methods: The broccoli seedlings were used as materials to amplify the specific thiolase-specific cDNA fragments by homologous sequence-based PCR. The 3 ’end and the 5’ end of cDNAs were amplified by 3’RACE and 5’RACE respectively. Broccoli glycosidase full-length cDNA sequence. The amino acid sequences encoded by the on-line protein analysis system were analyzed for their structural features, and the conserved domains were searched in the NCBICDD database. The on-line protein homology modeling was performed on the SWISS-MODEL and the NJ phylogenetic tree was constructed by using MEGA3.1. Results: The full length cDNA of this gene was 1830bp in length and contained a 1641bp ORF, 31bp 5 ’untranslated region and 158bp 3’ untranslated region. The gene was named BoMyr2 (GenBank Accession No. EU004075) Amino acid length signal peptide and nine asparagine N-terminal glycosylation sites, the molecular weight (Mw) of the encoded protein was about 62.2 kD and the isoelectric point (pI) was 8.71. BoMyr2 deduced that the amino acid sequence contained glycosyl hydrolase Family 1, a unique glycosyl hydrolase domain, encodes a protein with a similar three-dimensional structure to the white-glycerase beta-glucanase in the PDB library and has a pronounced β / α (TIM) barrel structure. BoMyr2 should be classified as a family of thiolase genes MB sub-family.