新型cry7Ab基因的鉴定克隆、表达与杀虫活性

本文应用PCR-RFLP法鉴定出Bt菌株HQ40中含有cry7Ab基因，并根据cry7A全长基因序列设计特异性引物，成功克隆了该基因。该基因核苷酸序列已经在国际基因库GeneBank中登记，其登录号为EU380678，并由Bt δ-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。生测结果表明：Cry7Ab4蛋白trypsin酶解液对鞘翅目的大猿叶甲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及293.79µg/ml。表达产物虽不能使鳞翅目的小菜蛾、甜菜夜蛾和亚洲玉米螟死亡，但对它们的生长发育有明显的体重抑制作用。另外对马铃薯甲虫以及榆蓝叶甲也有体重抑制作用，而对直翅目的东亚飞蝗无毒。

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

We identified for cry7Ab gene by PCR –RFLP from Bacillus thuringiensis HQ40.Then we designed a pair of special primer for full length DNA according to the published sequence of cry7A genes, successfully cloned it into Bt-E.coli shuttle vector pSTK and and transformed into acrystalliferous strain HD73cry-.The Bt engineering bacteria was named HD7Ab4.The gene had been registered in GenBank with accession number EU380678 and designated as the novel gene cry7Ab4 by International Nomenclature Committee of Bt.The SDS-PAGE showed that the cry7Ab gene could be expressed as 130 kD peptide.In addition, rhombohedral crystals was observed.Subsequently,we purified crystal proteins from Bt engineering bacteria HD7Ab4 and wild-type HQ40 respectively,we used the trypsin-activation of pro -endotoxins to do toxicity assay against a series of Orthopteran、 Lepidoptera、Coleoptera.The bioassay results indicated that the cry7Ab4 toxic protein had distinctly insecticidal activity against Colaphellus bowringi Baly.However,it exhibited little toxicity towards Oriental migratory locust ,Plutella xylostella、Spodoptera exigua、Ostrinia furnacalis、Pyrrhalta aenescens、Leptinotarsa decemlineate.