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【目的】Photorhabdus luminescens TT01基因组中的一对ORF plu4437-plu4436(简称pirA2B2)的预测氨基酸序列与另一对已证明编码产物有口服杀虫活性的ORF plu4093-plu4092(简称pirA1B1)有50%和45%的一致性,本文旨在研究pirA2B2基因座的表达产物是否也有杀虫活性。【方法】PCR扩增并克隆了pirA2,pirB2和pirA2B2基因,构建了重组表达载体pQE-pirA2,pQE-pirB2和pQE-pirA2B2并分别转入M15菌株表达,经SDS-PAGE和Western blot检测证明,3个重组菌株经IPTG诱导后,分别成功表达了可溶的PirA2,PirB2和PirA2B2蛋白。用亲和层析结合脱盐技术对3个重组菌株表达的外源蛋白分别进行纯化,并通过生物测定确定纯化蛋白的杀虫活性。【结果】生物测定结果显示联合表达的PirA2B2对大蜡螟和斜纹夜蛾五龄幼虫均有明显的血腔杀虫活性,LD50分别为每虫4.0和2.8μg,单独表达的PirA2或PirB2对上述2种害虫没有血腔杀虫活性,但两者的混合物具有与两者联合表达相似的杀虫活性;PirA2B2对大蜡螟和斜纹夜蛾初孵幼虫均无口服杀虫活性。【结论】pirA2B2是P.luminescens TT01菌株基因组中的另一个二元杀虫毒素基因。【意义】pirA2B2的成功克隆表达和杀虫功能的确定为进一步研究其与pirA1B1的关系以及该基因的表达调控等打下了基础。
【Objective】 The predicted amino acid sequence of a pair of ORF plu4437-plu4436 (referred to as pirA2B2) in Photorhabdus luminescens TT01 genome is 50% and 45% of that of ORF plu4093-plu4092 (pirA1B1), which has been shown to have oral insecticidal activity against the encoded product % Consistency, this paper aims to study whether the pirA2B2 locus expression product also has insecticidal activity. 【Methods】 The recombinant plasmids pQA-pirA2, pQE-pirB2 and pQE-pirA2B2 were amplified and cloned by PCR. The recombinant plasmids were transformed into M15 strain respectively. After induced by IPTG, three recombinant strains successfully expressed soluble PirA2, PirB2 and PirA2B2 protein, respectively. Purification of the foreign proteins expressed by the three recombinant strains was carried out by affinity chromatography and desalination respectively, and the biocidal activity of the purified protein was determined by bioassay. 【Result】 The results of bioassay showed that PirA2B2 co-expressed obvious blood-borne insecticidal activity against larvae of both Wolveria sp. And Spodoptera litura, with LD50 of 4.0 and 2.8 μg / L, respectively. PirA2 or PirB2, The two kinds of pests had no intracerebral insecticidal activity, but the mixture of the two had insecticidal activity similar to that of the two. PirA2B2 had no oral insecticidal activity against the larvae of the waxworm and Spodoptera litura. 【Conclusion】 pirA2B2 is another binary insecticidal toxin gene in the genome of P.luminescens TT01 strain. 【Significance】 The successful cloning, expression and insecticidal function of pirA2B2 laid the foundation for further study of its relationship with pirA1B1 and the regulation of its expression.