一种新的甘露糖结合凝集素——朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列。该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白。成熟蛋白由109个氨基酸残基组成,分子量为11.9kD。成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4％、85.3％、80.7％和83.5％的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY)。

Molecular Cloning of a Novel Mannose-binding Lectin Gene from Bulbs of Amaryllis vittata (Amaryllidaceae)

A new mannose-binding agglutinin gene was cloned from bulbs of Amaryllis vittata Ait. The fulllength cDNA of A. vittata agglutinin (AVA) was 686 bp. The start codon of ava cDNA was at 41-43 bp and the stop codon was at 515-517 bp. Analysis in the BLAST of GenBank showed that ava gene encodes a protein precursor composed of a signal peptide, mature protein and C-terminal amino acid cleavage sequence. The mature protein of AVA includes 109 amino acid residues and the molecular weight is 11.9 kD. The homologous analysis showed that the identity between AVA and Galanthus nivalis agglutinin, Narcissus hybrid cultivar agglutinin, Lycoris radiata agglutinin, Clivia miniata agglutinin are 73.4%, 85.3%, 80.7%, 83.5%, respectively. Molecular modeling of AVA indicated that its three-dimensional structure strongly resembles that of the snowdrop agglutinin. Blocks' analysis revealed that the deduced amino acid sequence of AVA has three functional domains specific for agglutination and three carbohydrate-binding boxes (QDNY).