韭莲凝集素基因的克隆及序列分析-作物遗传育种专业论文.docxVIP

韭莲凝集素基因的克隆及序列分析摘要 韭莲凝集素基因的克隆及序列分析 摘要 l近年来植物抗虫基因工程研究进展迅速，并分离出了不少的抗虫基因。 ＼ 然而：针对同翅目害虫的抗虫基因却很少被分离。大量的研究结果表明，植 物凝集素具有有效的抗虫作用，其中雪花莲外源凝集素(GNA，Ga]anthus nivalis agglutinin)已在植物基因工程中广泛应用，并对同翅目害虫如 蚜虫和褐飞虱等表现出较好的抗性·心 本实验采用cDNA末端快速扩增(Rapid amplification of cDNA ends， RACE)技术，首次从中国特有的野生植物韭莲(又名风雨花．Zephyranthes grandiflora)的叶片中分离克隆出了一个新的凝集索基因，对基因进行了序 列分析和功能预测，井成功地构建了韭莲凝集素基因的植物表达载体和原核 表达载体。为今后研究该基因的分子进化、功能表达及农作物的抗虫基因工 程等奠定了基础。惧体的实验结果如下： 1．基因克隆及序列分析：根据石蒜科(Amarylljdaceae)植物凝集素的 保守Ⅸ段，设计引物，采用RACE技术从韭莲中克隆出K度为952 bp的新凝 集素基因，包含了完摧的编码序列(576bp)。它编码一个全长191个氨基酸 的蛋白。与雪花莲外源凝集的相似性达59％，被命名为ZGA(Zephyrathes grindiflora agglutinin)。对该基因所推导出的氨基酸进行了信号肽剪 切位点分析、蛋向质结构域分析和专一结合糖基化分析，结果表明与其他石 蒜科种类的凝集素非常相似。推测此基冈具有与GNA相似的抗性功能。 2．植物表达载体的构建：在韭莲凝集素基冈(zga,Zephyrathes grandiflora agglutinin gene)克隆的基础上，构建了植物双元表达载体 p2300zga。并成功地导入到农杆菌EHAl05中。 3．原核表达载体的构建：将韭莲凝集素成熟蛋白的编码序列构建到原 核表达载体pET32a(+)中，井已经成功地转化到表达宿主苗BL21中。0 关键词：盎莲外源凝集素；基因克隆：RAcE：序列分析：载体构建 l瓣雌 些篷坚叁茎墨璺竺查竺垦生型坌堑 些篷坚叁茎墨璺竺查竺垦生型坌堑 一一一—— Abstract During the last few years important progress has been made in the study of plant anti-insect gene engineering，while a number of anti-insect genes were isolated and well documented．However,few genes resistant to homoptera (sap-sucking)insects were isolated．Recent studies have shown that some plant lectins from Amaryllidaceae species are toxic to sap—sucking insects in artificial diet assay,among which the lectin(GNA)from snowdrop(Galanthus nivalis)is the most toxic and has been most widely used in plant insect·resistant breeding． Transgenic plants expressing GNA showed significant insecticidal activity towards homoptera insects such as aphids and brown planthopper(BPH)in bioassay and feeding tests． In this paper a lectin gene was firstly isolated from leaves ofZephyranthes grand(flora，belonging to Amaryllidaceae，by the use of rapid amplification of cDNA ends(RACE)technique．Characterization，functional prediction and construction of plant and prokaryotic expression vector of this novel gene were studied．The analyzing results of t