对绿盲蝽具有杀虫活性Bt菌株的筛选及Cry15Aa蛋白活性的研究

摘要

Transgenic cotton with cry genes has controlled the main cotton pests (Helicoverpa armigera )effective-ly,however another non target mirid bugs (Heteroptera:Miridae)with sucking mouthparts have increased popu-lation sizes year by year.It has not been reported to the effective insecticidal genes of Bacillus thuringiensis a-gainst Apolygus lucorum (Meyer-Dür),in this study,Bt insecticidal genes and isolates stored in our laboratory were prepared to assay the insecticidal activity against A.lucorum .The cry 15Aa gene which belongs to Mtx in our lab was used to perform the bioassay to A.lucorum ,the result showed that Cry15Aa polypeptides was certain insecticidal activity against A.lucorum .After bioassay against A.lucorum ,twenty A.lucorum toxic Bt isolates were obtained.The LC-MS/MS identification showed these isolates might contains Cry1Aa,Cry1Ab,Cry1Ac, Cry1Ae,Cry1Af,Cry1Ag,Cry1Ah,Cry1Ba,Cry1Be,Cry8Ha and other proteins which are insecticidal to lepi-doptera and coleopteran pests,and four of them contains peptide fragment similar to Cry15Aa which belong to Mtx protein.But subsequence gene identification results indicated that these isolates not contains full length cry 15Aa gene,it suggested that these isolates contains new cry 15 type genes.The results indicated that these Bt i-solates may contain new insecticidal proteins toxic to mirid bugs.The obtaining of novel Bt isolates and the discovery of Cry15Aa with insecticidal activity against A.lucorum are significant to the biological control for A.lucorum .%Bt 棉有效控制了棉田主要害虫棉铃虫（Helicoverpa armigera ），然而原来处于次要地位的刺吸式口器害虫盲蝽（Heteroptera：Miridae）为害逐年加重，目前对绿盲蝽（Apolygus lucorum Meyer-Dür）有效的抗虫基因未见报道。本研究以本实验室保存的 Bt 杀虫基因和菌株为材料，对绿盲蝽进行杀虫活性筛选。利用本实验室先前克隆的Mtx 类杀虫基因 cry15Aa 表达产物进行绿盲蝽杀虫活性测定，研究结果显示 Cry15Aa 蛋白对绿盲蝽具有一定的杀虫活性。通过杀虫活性测定，获得对绿盲蝽有效的 Bt 菌株20株；对这些菌株表达蛋白进行质谱鉴定，LC-MS／MS质谱鉴定结果显示，这些菌株可能含有 Cry1Aa、Cry1Ab、Cry1Ac、Cry1Ae、Cry1Af、Cry1Ag、Cry1Ah、Cry1Ba、Cry1Be、Cry8Ha 等十余种对鳞翅目、鞘翅目害虫有杀虫活性的蛋白片段，并且有4株 Bt 菌株样品中检出了 Mtx 类杀虫蛋白 Cry15Aa 的片段。但是进一步基因鉴定结果表明，这4株菌株并不含有 cry15Aa 全长基因，推测这些菌株中含有 Cry15类的新基因。上述研究结果表明这些菌株中可能存在对盲蝽有效的新型杀虫蛋白。本研究在发现了 cry15Aa 基因所表达的蛋白对绿盲蝽具有杀虫活性的同时，获得了对绿盲蝽具有杀虫活性的 Bt 新菌株，对绿盲蝽的生物防治具有重要的意义。