抗鞘翅目δ-内毒素及毒素基因文库的构建

Coleopterancidal 6-endotoxin and Constructing Its Genomic Library

研究了对鞘翅目昆虫有毒效的5个苏云金芽孢杆菌新菌株YM-03、Sph04-04、YK14-01、SH11-05、Sph16-01伴孢晶体的蛋白质组成,以柳蓝叶甲为供试虫测定了它们的LC_(50)值,其中YM-03毒力最高。测定了YM-03晶体蛋白N-末端部分氨基酸序列。用琼脂糖凝胶电泳快速检测了5株菌的质粒组成,证明其质粒图型各不相同。以广谱的cosmid质粒pLAFRI为载体,通过限制酶EcoRI部分酶解获得“目的”DNA片段,构建了菌株YM-03的总DNA文库,对17个抗性克隆的质粒进行酶切分析表明,其中含有外源片段的克隆占总数的76％,超过要求的理论值。以人工合成的杀鞘翅目基因的18bp保守序列片段为探针,筛选了近1200个抗性克隆,获得了3个阳性克隆,LE392(PBYM2)、LE392(pBYM3)和LE392(pBYM4),毒力测定试验表明LE392(pBYM3)和LE392(pBYM4)有一定表达,表明其携带有δ-内毒素基因。

The composition of crytal protein and plasmid patterns, in tive new coleopterancidal strains of Bacillus turingiensis were investigated. Larvae of Plagiodera versicolra was chosen as model insect in assay of toxicity. Strain YM-03 had the highest toxicity. Plasmids patterns were detected, by rapid agarose gel electrophoresis. It was demonstrated that plasmid patterns of five strains were quite different. The partial amino acid sequences for N -terminal of YM-03 crystal protein was analyzed by amino acid analyzer. Genomic library of coleopterancidal S-endotoxin of YM-03 has been constructed. The EcoRI fragements (20-30kDa) of the tatol DNA were ligated with vector DNA of pLAFRI. Thirteen clones which contained both of pLAFRI and foreign DNA fragements out of 17 recombined plasmids were obtained. The frequency of recombinant clones was 76%. Three positive clones, named LE392(pBYM2) , LE392 (pBYM3) and LE392(pBYM4) were detected from 1200 resistant clones by using a synthesized 18bp probe from a coleopterancidal S-endotoxin gene. It was demonstrated that LE392 (pBYM3) and LE392(pBYM4) have the same EcoRI digested pattern,they all contained coleopteran specific S-endotoxin gene but showed a different level of expression.