School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Division of Entomology, Indian Agricultural Research Institute, New Delhi 110012, India.
Int J Biol Macromol. 2020 Sep 15;159:394-401. doi: 10.1016/j.ijbiomac.2020.05.078. Epub 2020 May 15.
Xenorhabdus nematophila strain ATCC 19061 is an insect pathogen that produces various protein toxins which intoxicate and kill its larval host. In the present study, we have described the cloning, expression and characterization of a 76-kDa chitinase protein of X. nematophila. A 1.9 kb DNA sequence encoding the chitinase gene was PCR amplified and cloned. Further, the chitinase protein was expressed in Escherichia coli and purified by using affinity chromatography. Two highly conserved domains were identified GH18 and ChiA. The purified chitinase protein showed chitobiosidase activity, β-N-acetylglucosaminidase and endochitinase activity, when enzyme activity was measured using respective substrates. The purified chitinase protein was found to be orally toxic to the larvae of a major crop pest, Helicoverpa armigera when fed to the larvae mixed with artificial diet. It also had adverse effect on the growth and development of the surviving larvae. Surviving larvae showed 9-fold reduction in weight, as a result the transformation of larvae into pupae was adversely affected. Our results demonstrated that the chitinase protein of X. nematophila has insecticidal property and can prove to be a potent candidate for pest control in plants.
嗜线虫致病杆菌（Xenorhabdus nematophila）菌株 ATCC 19061 是一种昆虫病原体，它会产生多种蛋白毒素，使幼虫宿主中毒并死亡。在本研究中，我们描述了嗜线虫致病杆菌的 76 kDa 几丁质酶蛋白的克隆、表达和特性。我们通过 PCR 扩增并克隆了编码几丁质酶基因的 1.9 kb DNA 序列。进一步，通过亲和层析法在大肠杆菌中表达并纯化了几丁质酶蛋白。鉴定出两个高度保守的结构域：GH18 和 ChiA。当使用各自的底物测量酶活性时，纯化的几丁质酶蛋白显示出几丁质酶活性、β-N-乙酰氨基葡萄糖苷酶和内切几丁质酶活性。当将纯化的几丁质酶蛋白与人工饲料混合喂养给幼虫时，发现其对主要作物害虫棉铃虫的幼虫具有口服毒性。它还对幸存幼虫的生长和发育产生了不利影响。幸存的幼虫体重减轻了 9 倍，因此幼虫转化为蛹的过程受到了不利影响。我们的研究结果表明，嗜线虫致病杆菌的几丁质酶蛋白具有杀虫特性，可作为植物害虫防治的有效候选物。
