Masson L, Préfontaine G, Péloquin L, Lau P C, Brousseau R
National Research Council of Canada Biotechnology Research Institute, Montreal, Quebec.
Biochem J. 1990 Jul 15;269(2):507-12. doi: 10.1042/bj2690507.
Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.
苏云金芽孢杆菌库尔斯塔克亚种的两种具有商业重要性的菌株（NRD - 12和HD - 1）各自包含三个序列部分相同的基因，这些基因编码三类130 - 135 kDa的原毒素（称为4.5、5.3和6.6原毒素），它们对各种鳞翅目幼虫具有毒性。这些基因产物结合形成细胞内双锥体P1晶体。从这两种菌株中分离出的每个基因都在大肠杆菌中进行了克隆和表达。在限制性内切酶水平上对克隆基因进行分析，结果显示在特定基因类别内的基因之间没有可检测到的差异。通过对纯化的P1晶体进行溴化氰裂解，并以纯化的重组基因产物作为参考蛋白，对两种菌株的P1晶体组成进行了定量分析。针对烟色卷蛾（CF - 1）昆虫细胞系的快速体外草坪细胞毒性试验为P1晶体中高含量6.6原毒素基因产物的存在提供了独立验证。结果表明，尽管NRD - 12或HD - 1的P1晶体中都存在所有三种基因产物，但两种晶体之间只有4.5和5.3原毒素的含量有所不同，而6.6原毒素的含量相似，在两种菌株中均约占P1晶体的三分之一。
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