雪花莲外源凝集素基因的克隆、序列分析和植物表达载体的构建  

Cloning and Sequencing of Snowdrop Lectin cDNA and Construction of Plant Expression Vector

作　　者：朱玉[1] 吴茜[1] 高越峰[1] 徐鸿林[1] 刘春明[1] 周兆斓 朱祯[1] 李向辉[1] 

机构地区：[1]中国科学院遗传研究所

出　　处：《农业生物技术学报》1997年第4期331-338,共8页Journal of Agricultural Biotechnology

基　　金："总理基金"资助;美国"洛克菲勒"资助

摘　　要：雪花莲外源凝集素对同翅目害虫有强烈的抑制作用，本研究从雪花莲叶片中分离出总ＲＮＡ，通过ＲＴ－ＰＣＲ方法扩增出了雪花莲外源凝集素基因，并将其克隆到ｐＢｌｕｅｓｃｒｉｐｔＫＳ（＋）的ＥｃｏＲⅤ位点。酶切图谱及ＤＮＡ序列分析表明克隆的片段长度为４８４ｂｐ，包含了完整的外源凝集素的编码序列。编码多肽由１５７个氨基酸组成，其中包括Ｎ－端２３个氨基酸组成的信号肽序列，１０５个氨基酸组成的成熟蛋白序列和２９个氨基酸组成的Ｃ－端序列。我们从多个克隆中筛选出一个与报道序列最为接近的克隆，它们之间核苷酸序列及推导出的氨基酸序列的同源性分别为９６％和９４％，我们用这个克隆构建了植物表达载体，并用于水稻和小麦的转化。Snowdrop lectin (Galanthus nivalis agglutinin, GNA) is a kind of protein which can add protection against diptera insect pests effectively. The GNA gene was amplified from total snowdrop leaves RNA by RTPCR, and was cloned into EcoRⅤ site of pBluescript KS(+). DNA sequence analysis indicated that the cloned 484 bp DNA fragment carry the entire GNA coding sequence, which encoded a peptide consisted of 157 amino acids, including 23 amino acid signal peptide sequence at N end, 105 amino acid mature protein and a Cterminal extension of 29 amino acids. We selected a clone pGNA14 which has the highest homology compared with the published data, showing 96% and 94% homology at the nucleotide and at the deduced amino acid sequence respectively. A GNA plant expression vector has been constructed for rice and wheat transformation. 

关 键 词：雪花莲 外源凝集素 DNA序列 抗虫基因 

分 类 号：S332.3[农业科学—作物遗传育种]