掌叶半夏凝集素基因的克隆及亚细胞定位分析

Genomic Cloning an Agglutinin Gene from Pinellia pedatisecta and Analysis on Its Subcellular Localization

为了克隆掌叶半夏凝集素基因(Pinellia pedatisecta agglutinin,PPA),并对其进行生物信息学分析和亚细胞定位。采用PCR技术扩增掌叶半夏凝集素基因的ORF序列,构建pCAMBIA1300-35S-PPA-e GFP重组载体,导入农杆菌GV3101并在烟草中进行瞬时表达,采用激光扫描共聚焦显微镜观察其亚细胞定位。克隆获得的PPA基因全长由777个核苷酸组成,编码258个氨基酸,含有3个甘露糖结合位点和2个保守的B-lectin结构域,Gen Bank登录号为KF154981,氨基酸序列登录号为AGV40779。序列比对表明,该序列与天南星、滴水珠、花南星和半夏凝集素的氨基酸序列相似性分别为96%,91%,86%和83%。亚细胞定位的结果表明,pCAMBIA1300-35S-PPA-e GFP融合蛋白在细胞核内无分布,而是点状分布在细胞质膜上。为进一步解析掌叶半夏凝集素基因的结构与功能,开展掌叶半夏凝集素抗病虫害机制的研究奠定基础。

To clone Pinellia pedatisecta agglutinin gene（PPA）from the leaves of Pinellia pedatisecta,analyzed on its biological information characteristics,and observed its subcellular localization.The open reading frame sequence of PPA gene was amplified by PCR method.The recombinant vector pCAMBIA1300-35S-PPA-eGFP was constructed and then transferred into Agrobacterium strain GV3101.Agrobacterium suspensions expressing PPA were injected at different concentrations into Nicotiana tabacum leaves and the subcellular location was observed by confocal laser scanning microscope.It showed that the open reading fragment sequence length of PPA gene was 777 bp,encoding 258 amino acids with GenBank accession number KF154981 and AGV40779,respectively.It contained two conservative B-lectin domains and three mannose binding sites.Sequence alignment demonstrated that the deduced amino acid sequence of PPA,which were 96%,91%,86% and 83% identical respectively,with the corresponding region of Arisaema heterophyllum, Pinellia cordata,Arisaema lobatum and Pinellia ternata.The subcellular location results indicated that the PPA-eGFP was located in the plasma membrane as large spots,but not found in nucleus.The results would provide the foundation for further study the structure and function of PPA gene,and elucidate anti-microbial mechanism of PPA.