苏云金芽孢杆菌伴孢晶体中20-kb DNAs上染色体基因的鉴定

已有的研究表明苏云金芽孢杆菌(Bacillus thuringiensis,Bt)4.0718菌株菱形晶体中含有与原毒素紧密结合的20-kb DNAs,利用PCR-RFLP分析发现在这些20-kb DNAs上含有cry1Aa、cry1Ac、cry2Aa和cry2Ab基因.本研究利用特异引物从4.0718菌株中20-kb DNAs上分别扩增出带有自身调控序列的cry1Ac和cry2Aa基因,并将其分别电转至Bt无晶体菌株Cry-B中获得重组菌株Cry-B(pHC42)和Cry-B(pHC39).SDS-PAGE和透射电镜分析表明这两种重组菌株能够分别产生由130-kDa的Cry1Ac原毒素形成的菱形晶体以及由65-kDa的Cry2Aa原毒素形成的方形晶体.毒力生测的结果显示,这两种菌株的表达产物对棉铃虫(Helicoverpa armigera)幼虫具有预期的杀虫效果.重组菌株分别形成的两种晶体经不同的缓冲液溶解后能释放出与20-kb DNAs紧密结合的原毒素,同时在20-kb DNAs上均可检测到源于Bt染色体的spo0A和nprA基因片段.

Identification of Chromosomal Genes on 20-kb DNAs from Parasporal Crystal of Bacillus thuringiensis

Our previous study reported that 20-kb DNA fragments were intimately associated with Cry1 bipyramidal crystal of Bacillus thuringiensis strain 4.0718 and cry1Aa,cry1Ac,cry2Aa,and cry2Ab genes were detected on the 20-kb DNAs through PCR-RFLP.In the present study,cry1Ac and cry2Aa genes with their own regulatory sequence were amplified from these 20-kb DNAs using specific primers.The two genes were electroporated into an acrystalliferous Bacillus thuringiensis strain Cry-B,producing recombinant strains Cry-B(pHC42) and Cry-B(pHC39) respectively.SDS-PAGE and transmission electron microscopy analyses showed that the two recombinant strains could produce bipyramidal crystal of 130-kDa Cry1Ac protoxin and cuboidal crystal of 65-kDa Cry2Aa protoxin.Bioassay results proved that crystal-spore mixture from the recombinants was toxic to Helicoverpa armigera.After being solubilized by different buffer,the bipyramidal and cuboidal crystals both released protoxin-20-kb DNA complex,and the chromosome-specific spo0A and nprA genes could be detected on these DNA fragments.