利用末龄幼虫蜕和蛹壳对单头昆虫基因的无损伤检测-应用昆虫学报.DOCVIP

利用末龄幼虫蜕和蛹壳对单头昆虫基因的无损伤检测* 郑美艳** 朱冠恒 陈 严 韩 琪 杨亦桦 董双林*** （南京农业大学植保学院昆虫学系，南京 210095） 摘 要 【目的】【】3种鳞翅目昆虫斜纹夜蛾Spodoptera litura Fabricius、二点委夜蛾Athetis lepigone M?schler和小菜蛾Plutella xyllostella Linnaeus，收集末龄幼虫蜕及蛹壳，利用常规分子生物学技术进行基因组DNA提取、靶标基因PCR扩增、琼脂糖凝胶电泳检测、连接转化和单克隆测序验证。【】DNA，以其为模板对GOBP1基因进行PCR扩增，产物经琼脂糖凝胶电泳检测得到单一、明显的条带，进一步经连接转化和单克隆测序得到目的序列；但由于小菜蛾末龄幼虫蜕和蛹壳太小，以同样方法提取的基因组DNA浓度太低，PCR产物经电泳检测，未能得到目的条带。【】DNA，通过常规PCR技术克隆特定基因序列，为突变品系筛选过程中昆虫个体的无损伤基因型检测提供了方法。 关键词DNA，PCR，突变品系 A non-destructive method of genotyping individual insects from the exuviate of final instar larvae, or puparia ZHENG Mei-Yan** ZHU Guan-Heng CHEN Yan HAN Qi YANG Yi-Hua DONG Shuang-Lin*** (Department of Entomology, Nanjing Agricultural University, Nanjing 210095, China) Abstract [Objectives] The single pair mating strategy is often used to screen homozygous insect strains for the functional study of insect genes. This requires genotyping individual insects without killing, or otherwise harming them, before pair combinations can be made. The purpose of this study was to evaluate a non-destructive method of genotyping individual insects using the exuviate of final instar larvae, or puparia. [Methods] Genomic DNA was extracted from exuviates of the final instar larvae and puparia of Spodoptera litura Fabricius, Athetis lepigone M?schler and Plutella xyllostella Linnaeus with DNAiso reagent. The general odorant binding protein 1 (GOBP1) gene was used as a representative gene and amplified by PCR, followed by agarose gel electrophoresis, ligation, transformation, and TA clone sequencing. [Results] Agarose gel electrophoresis of GOBP1 PCR products from S. litura or A. lepigone produced a bright, single band of the expected size, the identity of which was confirmed by TA cloning and sequencing. However, due to the low quantity of larval exuviate and small size of puparia, the concentration of DNA from P. xyllostella was very low and the expected target band of GOBP1 was not detected by gel electrophores