海芋凝集素cDNA的分子克隆及其性质预测

摘要

用cDNA末端快速扩增-聚合酶链式反应(RACE-PCR)方法克隆了海芋(Alocasia macrorrhiza)凝集素的全长cDNA(GenBank检索号DQ340864),并用多种生物信息学工具对其性质进行了预测.根据来源于天南星科其他植物的凝集素和类似蛋白的保守区的DNA序列,设计了几个海芋凝集素基因aml特异引物(GSP).用RNeasy试剂盒从海芋块茎中提取出总RNA,并以此为模板,用SMARTTM RACE cDNA扩增试剂盒提供的经特殊设计的通用引物以及不同的基因特异引物,分别获得海芋凝集素5'-和3'-RACE-PCR扩增片段.这些PCR产物经0.8%琼脂糖凝胶纯化后,分别与T克隆载体pMD 18-T相连,筛选获得阳性克隆并提取质粒,经双酶切和特异引物的PCR验证无误后,进行序列分析.从5'-和3'-RACE-PCR测序结果拼接出全长海芋凝集素cDNA序列,并用新设计自5'-RACE-PCR 5'末端的引物GSP7进行全长3'-RACE-PCR反应,获得全长海芋凝集素cDNA克隆并再次测序验证.这一新克隆的海芋凝集素cDNA的长度为1124核苷酸,分析表明它是一个编码270个氨基酸残基的蛋白质,其等电点为pH 5.7,相对分子量为29.7 kD.同源性分析结果表明,海芋凝集素与其他来源于天南星科的甘露糖凝集素以及相似蛋白具有高度同源性.在海芋凝集素序列中发现了2个B型凝集素功能区域和3个甘露糖的结合位点.综合上述信息,认为这一新克隆的海芋凝集素cDNA是一个编码甘露糖识别凝集素的基因序列.%The cDNA of Alocasia macrorrhiza lectin(aml, GenBank accession number: DQ340864) was cloned by RACE-PCR and its characteristics were predicted by various bioinformatics tools. GSPs (Gene Specific Primers) were designed according to the conserved regions of the genes encoded for lectins and similar proteins from the same family Araceae. Total RNAs were extracted from the tubers of A. macrorrhiza by Qiagen RNeasy mini kit. The 3'- and 5'-RACE-PCRs were performed with the isolated total RNAs by SMARTTM RACE cDNA amplification kit from BD Biosciences Clontech Company, respectively. The purified PCR products were ligated with pMD 18-T vector, and the confirmed clones were sequenced. The full-length cDNA of aml was obtained by combination of 3'- and 5'-end sequences, and was then confirmed by full-length 3'-RACE-PCR. The aml cDNA is 1 124 bp long. The deduced amino acid length of AML lectin is 270 aa. Its relative molecular weight is 29.7 kD. The results of homologous analysis showed a high similarity between AML and other mannose-binding lectins and similar proteins from Araceae family. Two typical B-lectin domains and three mannosebinding motifs were found in the sequence of AML. With all these taken together, it can be concluded that this newly cloned aml cDNA encodes for a mannose-binding lectin.