新型cry7Ab基因的鉴定、克隆、表达与杀虫活性  

Identification,Cloning and Expression for Novel cry7Ab Gene and Its Insecticidal Activity

作　　者：邓淑[1,2] 束长龙[2] 林毅[1] 宋福平[2] 张杰[2] 

机构地区：[1]华侨大学化工学院生物工程与技术系,厦门361021 [2]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100193

出　　处：《农业生物技术学报》2009年第5期908-913,共6页Journal of Agricultural Biotechnology

基　　金：植物病虫害生物学国家重点实验室开放基金(No.2007IP7);国家高技术研究与发展计划项目(No.2006AA10A212和No.2006AA02Z189)资助

摘　　要：应用PCR-RFLP法鉴定出苏云金芽胞杆菌(Bacillus thuringiensis,Bt)HQ40中含有cry7Ab基因,并根据cry7A全长基因序列设计特异性引物,成功地克隆了该基因。该基因核苷酸序列已在GenBank中登录,登录号为EU380678,并由Btδ-内毒素基因国际命名委员会正式命名为cry7Ab4。通过穿梭载体pSTK将该基因导入Bt无晶体突变株中,获得工程菌HD7Ab4。SDS-PAGE分析表明,cry7Ab4基因在其中能正常表达,并形成菱形晶体。提取工程菌HD7Ab4和野生菌HQ40晶体蛋白,并在体外用胰蛋白酶酶解活化。分别对直翅目、鳞翅目和鞘翅目的害虫进行了杀虫活性测定。结果表明,Cry7Ab4蛋白trypsin酶解液对鞘翅目的大猿叶甲(Colophellus bowringi)显示了一定的杀虫活性,其野生菌蛋白及表达蛋白酶解液LC50分别为231.59μg/mL和293.79μg/mL。但对鳞翅目的小菜蛾(Plutella xylostella)、甜菜夜蛾(Spadoptera exigua)、亚洲玉米螟(Ostrinia fur-nacalis)、马铃薯甲虫(Leptinotarsa decemlineata)、榆蓝叶甲(Pyrrhalta aenescens)和直翅目的东亚飞蝗(Oroental migratory locust)基本无杀虫活性。Bacillus thuringiensis HQ40 was identified to harbour cry7Ab gene by PCR-RFLP.The full-length cry7Ab gene was amplified, cloned into Bt-Esceriehia coli shuttle vector pSTK, and then transformed into acrystalliferous Bt strain HD73cry-, which was named HD7Ab4.The gene was registered in GenBank with accession number EU380678 and was designated as a novel gene, cry7Ab4, by International Nomenclature Committee of Bt.The Cry7Ab4 protein was expressed as rhombohedral crystals.The crystal proteins of HD7Ab4 and HQ40 were prepared and processed by trypsin, and were then assayed against insect pests from Orthopteran, Lepidoptera and Coleoptera.The trypsin-processed crystal proteins of HQ40 and the trypsin-processed Cry7Ab4 protein had insecticidal activity against Colaphellus bowringi, with LC50 231.59 and 293.79 μg/mL, respectively.However, they exhibited little toxicities towards Oriental migratory locust, Plutella xylostella,Spodoptera exigua,Ostrinia furnacalis,Pyrrhalta aenescens,and Leptinotarsa decemlineata.

关 键 词：苏云金芽胞杆菌 cry7Ab4基因 克隆表达 杀虫活性 

分 类 号：S188[农业科学—农业基础科学]