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利用末龄幼虫蜕和蛹壳对单头昆虫 基因的无损伤检测
A non-destructive method of genotyping individual insects from the exuviate of final instar larvae, or puparia
郑美艳** 朱冠恒 陈 严 韩 琪 杨亦桦 董双林***
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作者单位:南京农业大学植保学院昆虫学系,南京 210095
中文关键词:幼虫蜕,蛹壳,基因组DNA,PCR,突变品系
英文关键词: larval exuviate, puparium, genomic DNA, PCR, mutant strain
中文摘要:

【目的】 在昆虫基因功能等相关研究中,通常需要利用单对交配策略来筛选纯合突变品系,如何在配对前确定个体基因型同时又不对昆虫造成损伤,显得尤为重要。本文旨在探讨利用末龄幼虫蜕和蛹壳进行单头昆虫的无损伤基因检测方法。方法 针对大小不同的3种鳞翅目昆虫斜纹夜蛾Spodoptera litura Fabricius二点委夜蛾Athetis lepigone Möschler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。结果 从斜纹夜蛾、二点委夜蛾末龄幼虫蜕和蛹壳提取的基因组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。结论 对于与斜纹夜蛾和二点委夜蛾相近或更大的昆虫,可以利用单头末龄幼虫蜕或蛹壳提取基因组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。

英文摘要:

[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 Möschler 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 electrophoresis. [Conclusion]  Insects of the same size, or larger, than S. litura and A. lepigone can be non-destructively genotyped by PCR using final instar larvae exuviate, or puparia.

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