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本研究将高分辨熔解分析(high resolution melting assay,HRMA)的方法应用于cry1I类基因的分型。根据已报道的cry1I类基因的保守区设计一对扩增片段为131 bp的通用鉴定引物,利用高分辨熔点仪对产物的熔解曲线进行分析。利用此方法对本实验室保存的标准菌株HD-12进行了cry1I类基因的鉴定,克隆到2个新型的cry1Ib和cry1Id基因,被Bt国际命名委员会分别正式命名为cry1Ib11和cry1Id2。这两种基因在E.coli Rosetta菌株中能正常表达81 kDa的蛋白。Cry1Ib11和Cry1Id2蛋白对小菜蛾Plutellaxylostella二龄幼虫和亚洲玉米螟Ostrinia furnacalis初孵幼虫具有显著的杀虫活性,LC50分别为6.13μg.mL 1和6.10μg.mL 1及11.18μg.mL 1和1.43μg.mL 1。但对甜菜夜蛾Spodoptera exigua及鞘翅目的大猿叶甲Colaphellus bowringi均没有明显毒杀作用。高分辨溶解分析法为新型cry基因的快速、准确鉴定提供了有效的工具。
In this study, high-resolution melting assay (HRMA) method was applied to cry1I-type genotyping. Based on the reported conserved regions of cry1I genes, a pair of universal primers was designed to amplify the fragment of 131 bp. The melting curve of the product was analyzed by high resolution melting point instrument. Using this method, the cry1I gene was identified in the standard strain HD-12 preserved in our laboratory. Two new cry1Ib and cry1Id genes were cloned and named by the Bt International Nomenclature Committee as cry1Ib11 and cry1Id2 respectively. Both of these genes normally express 81 kDa protein in E. coli Rosetta strain. The Cry1Ib11 and Cry1Id2 proteins have significant insecticidal activities against the larvae of Plutella xylostella second instar larvae and Ostrinia furnacalis larvae of the diamondback moth Plutella xylostella with LC50 of 6.13μg.mL 1 and 6.10μg.mL 1 and 11.18μg.mL 1 and 1.43μg .mL 1. However, there was no obvious toxic effect on Spodoptera exigua and Colephellus bowringi. High-resolution lysis assay provides an effective tool for the rapid and accurate identification of novel cry genes.