忽地笑α-葡萄糖苷酶编码基因的分子克隆与原核表达

Molecular Cloning and Prokaryotic Expression of α-Glucosidase Encoding Gene from Lycoris aurea

利用RACE技术从忽地笑(Lycoris aurea(L’Hér.)Herb.)花瓣中克隆获得一个α-葡萄糖苷酶编码基因Lau Agl。该基因cDNA全长2 810 bp,开放阅读框为2 613 bp,编码含有870个氨基酸残基的LauAgl蛋白质。LauAgl蛋白质氨基酸序列具有α-葡萄糖苷酶特征性的保守结构域和天冬氨酸残基组成的活性中心,其N端含有一个由21个氨基酸组成的信号肽序列,还具有一些蛋白质糖基化的潜在位点(N-X-S/T共有序列)。LauAgl蛋白质与石刁柏、椰子、油棕等其他植物的α-葡萄糖苷酶的氨基酸序列一致性达到63%以上。利用pET表达系统,通过截掉其N-端信号肽序列,可以实现LauAgl蛋白质在大肠杆菌中的诱导表达。本研究为Lau Agl蛋白质的功能鉴定及其在忽地笑种子萌发与生长发育中的作用研究提供理论基础。

In this stud y,aα-glucosidase encoding gene LauAgl was cloned from the petals of Lycoris aurea(L'Hér.)Herb.by using RACE technology.The full-length cDNA of LauAgl was 2 810 bp with a 2 613 bp open reading frame,encoding a LauAgl protein containing 870 amino acids residues.LauAgl protein amino acid sequence contained the active center composed of conservative domain and aspartic acid residue characterized byɑ-glucosidase,the N-terminal of which contained a signal peptide sequence consisting of 21 amino acids,and also had some potential sites(N-X-S/T common sequence)for possible glycosylation.The amino acid sequence of LauAgl protein shared more than 63%consistent with that ofα-glucosidases from Asparagus officinalis L.,Cocos nucifera L.,Elaeis guineensis Jacq.and other plants.Based on the pET expression system,LauAgl protein was successfully induced to express in Escherichia coli by truncating N-terminal signal peptide sequence.This study would provide a theoretical basis for the functional identification of LauAgl protein and its role in germination,growth and development of Lycoris aurea seeds.