稻胚凝集素基因的克隆、序列分析及表达  

Cloning,sequencing and haracterizationN of rice germ lectin and its expressing in Escherichia coli

作　　者：郝峥嵘[1,2,3] 刘庆法[1,2,3] 季昀[1,2,3] 叶鸣明[1,2,3] 陈永青[1,2,3] 沈大棱[1,2,3] 

机构地区：[1]复旦大学生命科学学院 [2]新疆农业科学院核生物研究所 [3]西北农业大学农学系

出　　处：《西北植物学报》1999年第2期183-189,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：上海市科委科学与技术发展基金

摘　　要：以水稻基因组ＤＮＡ为模板，以特异引物经聚合酶链式反应方法扩增出稻胚凝集素基因并克隆到Ｅ．ｃｏｌｉ质粒ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）的ＳｍａⅠ位点。序列分析表明，克隆到的基因片段大小为７８１ｂｐ，没有内含子，编码１条长２２７个氨基酸、分子量约２３ｋＤ的肽链，其中Ｎ－端２８个氨基酸是信号肽。与报道的稻胚凝集素ｃＤＮＡ序列进行顺序同源性比较，发现它们之间有很高的同源性（９９．７４％），其编码区第１６７和５６３位各有１个碱基突变，但两者均为同义突变，并未造成氨基酸序列的变化。回收插入片段克隆到表达载体ｐＲＳＥＴ－Ｃ中，在大肠杆菌ＢＬ２１（ＤＥ３）中得到表达，ＳＤＳ－ＰＡＧＥ电泳图谱扫描结果表明，融合蛋白表达量约占菌体总蛋白的５％。鉴于凝集素可能具有抗病虫害的能力，稻胚凝集素基因的克隆及其在原核系统中的成功表达有助于我们进一步揭示稻胚凝集素在植物防御以及其他生理活动中的功能。By the aid of Polymerase Chain Reaction,the sequence coding for rice germ lectin (RGL) was isolated and cloned into the Sma Ⅰ site of pBluescript SK (+).The sequence data showed that the cloned fragment is 781 bp.Like most plant lectin genes,there is no intron in the amplified sequence.The open reading frame of the fragment codes a 23 kD polypeptide which contains 227 amino acid residues and there is an signalsequence with 28 amino acid residues at its NH 2 terminus.The amplified fragment is highly homologous to the published cDNA sequence with similarity of 99.74%,having two base changes at 167 and 563 but without changing amino acid.The fragment was then expressed in E.coli strain BL21 (DE3) by inserting it into the expression vetor pRSET C.The expressed protein account for about 5% of the total soluble proteins.The establishment of this expression system provides an ideal model for further studies of its biological activities in vitro ,especially its defensive functions to pests and diseases and could be taken as a useful gene for plant genetic engineering.

关 键 词：稻胚凝集素基因 序列分析 基因表达 水稻 

分 类 号：S511.01[农业科学—作物学]