【摘要】 研究和利用不同苏云金芽孢杆菌(Bacillusthuringiensis,Bt)杀虫蛋白之间的增效作用是构建高效遗传工程杀虫剂、预防和延缓害虫抗性的重要途径。BtCry1Ab,Cry1Ac和Cry2Aa蛋白对重要的鳞翅目农业害虫具有高毒力,质粒pHT-BSK含有能在大肠杆菌(Escherichiacoli)-枯草芽孢杆菌(B.subtilis )有效表达的生物安全性标记基因卡那霉素抗性基因。将含Btcry1Ab,cry1Ac和cry2Aa全长基因的Bam HⅠ/PstⅠ、Bam HⅠ/XhoⅠ和SmaⅠ(BamHⅠ)/HindⅢDNA片段与pHT-BSK相应酶切产物连接,获得了单价cry/kmr组合的重组质粒pBlue-1Ab-kmr、pBlue-1Ab和pHT-2Aa;cry1Ab,cry1AcDNA片段与pHT-2Aa相应酶切产物相连接获得了Bt双价基因/kmr组合重组质粒pBlue-cry1Ab-kmr-2Aa和pBlue-cry1Ac-kmr-2Aa,重组质粒中所有cry基因与kmr均形成特异的BamHⅠ片段。限制酶切电泳分析和特异PCR扩增证实了重组质粒的准确连接。含有重组质粒的大肠杆菌显示了对小菜蛾(Plutellaxylostella )和玉米螟(Ostriniafurnacalis )的杀虫活性,对小菜蛾双价基因毒力高于单价基因,但对玉米螟它们之间没有明显差异。研究为进一步阐明Btcry2Aa与cry1Ab和cry1Ac之间在不同微生物细胞中的共表达及其表达产物之间的相互作用和构建双价基因杀虫工程菌株创造了条件。更多还原

【Abstract】 Research and application of synergism among different Bacillus thuringiensis (Bt ) insecticidal crystal proteins (Bt ICPs) is one of the most potential ways for construction of high toxic engineered strains. Cry1Ab,Cry1Ac and Cry2Aa protein from Bt are toxic to certain important lepidoptera insect pests. Plasmid pHT-BSK, which carried the kanamycin resistant gene kmr regarded as a biosafety resistant marker in genetic engineering and expressed both in Escherichia coli and Bacillus subtilis, was digested with Bam HⅠ/PstⅠ, Bam HⅠ/XhoⅠ and SmaⅠ/HindⅢ respectively. Full length cry1Ab,cry1Ac and cry2Aa genes in Bam HⅠ/PstⅠ, Bam HⅠ/XhoⅠand SmaⅠ/HindⅢ DNA fragments were ligated with the digested pHT-BSK respectively, and the recombinant plasmids containing cry gene/kmr:pBlue-1Ab-kmr, pBlue-1Ac-kmr and pHT-2Aa were obtained, and the dual cry-gene plasmids pBlue-cry1Ab-kmr-2Aa and pBlue-cry1Ac-kmr-2Aa were constructed by inserting the cry1Ab and cry1Ac fragments into the plasmid pHT-2Aa. In all recombinant plasmids, the cry and the kmr genes were located in the unique Bam HⅠ fragments, and the recombinant vectors were convinced with PCR and restriction analysis.The results of insecticidal bioassay indicated that the dual-cry- gene recombination was much more toxic than that of the mono- cry- gene to the Plutella xylostella larvae, but no obvious difference to the Ostrinia furnacalis larvae. This work laid a foundation for further research of the co-expression cry2Aa and cry1Ab or cry1Ac gene in the other bacterial cells and the synergism or inhibition relation among them.更多还原