苏云金芽孢杆菌中几丁质酶编码基因的表达及工程化苏云金芽孢杆菌日本亚种对舞毒蛾幼虫的毒性
Expression of chitinase-encoding genes in Bacillus thuringiensis and toxicity of engineered B. thuringiensis subsp. aizawai toward Lymantria dispar larvae.
作者信息
Lertcanawanichakul Monthon, Wiwat Chanpen, Bhumiratana Amaret, Dean Donald Harry
机构信息
Institute of Allied Health Sciences and Public Health, Walailak University, Thasala District, Nakhon Si Thammarat 80160, Thailand.
出版信息
Curr Microbiol. 2004 Mar;48(3):175-81. doi: 10.1007/s00284-003-4119-0.
Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively. Both plasmids were introduced into various strains of B. thuringiensis by electroporation. Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B. thuringiensis subsp. aizawai when grown under nonselective conditions. The production of chitinase from B. thuringiensis subsp. aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl beta-D- N,N'- diacetylchitobioside as the substrate. Moreover, B. thuringiensis subsp. aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate. In addition, B. thuringiensis subsp. aizawai harboring pHYB43 was more toxic to gypsy moth larvae ( Lymantria dispar) than parental B. thuringiensis subsp. aizawai or its clone harboring pHYA2.
将嗜水气单胞菌和环状芽孢杆菌4.1的几丁质酶基因克隆到质粒pHY300PLK中，分别命名为pHYA2和pHYB43。通过电穿孔将这两种质粒导入苏云金芽孢杆菌的各种菌株。质粒pHYB43在结构上通常是稳定的，但在非选择性条件下生长时，在苏云金芽孢杆菌 aizawai亚种中，其分离稳定性低于pHYA2。以4-甲基伞形酮基-β-D-N,N'-二乙酰壳二糖为底物，通过天然聚丙烯酰胺凝胶电泳可检测到携带pHYB43或pHYA2的苏云金芽孢杆菌 aizawai亚种几丁质酶的产生。此外，以乙二醇几丁质为底物，通过比色法测定，携带pHYB43的苏云金芽孢杆菌 aizawai亚种产生的几丁质酶活性比携带pHYA2的菌株高15倍。此外，携带pHYB43的苏云金芽孢杆菌 aizawai亚种对舞毒蛾幼虫（Lymantria dispar）的毒性比亲本苏云金芽孢杆菌 aizawai亚种或携带pHYA2的其克隆体更强。
Zotero 插件