Directorate of Oilseeds Research, ICAR, Rajendranagar, Hyderabad 500030, India.
Mol Biotechnol. 2013 Jul;54(3):795-802. doi: 10.1007/s12033-012-9627-3.
A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.
从当地有潜力的分离株 DOR Bt-1 中克隆了一个新的 cry1Ab 基因，该分离株是从蓖麻夜蛾（Achaea janata L.）尸体中分离得到的苏云金芽孢杆菌（Bacillus thuringiensis）菌株，来源于蓖麻（Ricinus communis L.）田。克隆的 cry1Ab 基因的核苷酸序列表明，开放阅读框由 3465 个碱基组成，编码一个由 1155 个氨基酸残基组成的蛋白质，估计分子量为 130kDa。同源性比较表明，cry1Ab 的推导氨基酸序列与 NCBI 数据库中已知的 Cry1Ab 蛋白有 7 个氨基酸残基的差异，该基因已被苏云金芽孢杆菌 δ-内毒素命名委员会指定为 cry1Ab26。cry1Ab26 被克隆到 pET 29a(+)载体中，并在 IPTG 诱导下由 T7 启动子控制在大肠杆菌 BL21 (DE3) 中表达。ELISA、SDS-PAGE 和 Western blot 分析证实了 130kDa 蛋白的表达。用棉铃虫幼虫进行的昆虫生物测定表明，部分纯化的 Cry1Ab26 在喂食后 5 天内导致 97%的死亡率。
