Hire Ramesh S, Makde Ravindra D, Dongre Tanaji K, D'souza Stanislaus F
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India.
Curr Microbiol. 2008 Dec;57(6):570-4. doi: 10.1007/s00284-008-9244-3. Epub 2008 Sep 16.
An indigenously isolated strain of Bacillus thuringiensis subsp. kenyae exhibited toxicity against lepidopteran as well as dipteran insects. The lepidopteran active cry1Ac protoxin gene coding sequence of 3.5 kb from this strain was cloned into vector pET28a(+). However, it could not be expressed in commonly used Escherichia coli expression hosts, BL21(DE3) and BL21(DE3)pLysS. This gene is classified as cry1Ac17 in the B. thuringiensis toxic nomenclature database. The coding sequence of this gene revealed that it contains about 3% codons, which are not efficiently translated by these expression hosts. Hence, this gene was expressed in a modified expression host, Epicurian coli BL21-Codonplus (DE3)-RIL. The expression of gene yielded a 130-kDa Cry1Ac17 protein. The protein was purified and its toxicity was tested against economically important insect pests, viz., Helicoverpa armigera and Spodoptera litura. LC(50) values obtained against these insects were 0.1 ng/cm(3) and 1231 ng/cm(2), respectively. The higher toxicity of Cry1Ac17 protein, compared to other Cry1Ac proteins, toward these pests demonstrates the potential of this isolate as an important candidate in the integrated resistance management program in India.
一株本土分离的苏云金芽孢杆菌肯尼亚亚种菌株对鳞翅目和双翅目昆虫均表现出毒性。将该菌株中3.5 kb的鳞翅目活性cry1Ac原毒素基因编码序列克隆到载体pET28a(+)中。然而，它无法在常用的大肠杆菌表达宿主BL21(DE3)和BL21(DE3)pLysS中表达。该基因在苏云金芽孢杆菌毒素命名数据库中被归类为cry1Ac17。该基因的编码序列显示，它含有约3%的密码子，这些表达宿主不能有效地翻译这些密码子。因此，该基因在一种改良的表达宿主，即艾氏大肠杆菌BL21-Codonplus (DE3)-RIL中表达。该基因的表达产生了一种130 kDa的Cry1Ac17蛋白。该蛋白被纯化，并对重要经济害虫棉铃虫和斜纹夜蛾进行了毒性测试。针对这些昆虫获得的LC(50)值分别为0.1 ng/cm(3)和1231 ng/cm(2)。与其他Cry1Ac蛋白相比，Cry1Ac17蛋白对这些害虫具有更高的毒性，这表明该分离株在印度综合抗性管理计划中作为重要候选菌株的潜力。
