草地贪夜蛾听觉基因Cut和Spalt时空表达分析及其功能研究
Spatio-temporal expression and function of the Spodoptera frugiperda Cut and Spalt auditory genes
何昕峪，吴 宇，王慧敏，张爱兵
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DOI：10.7679/j.issn.2095-1353.2023.111
作者单位：首都师范大学生命科学学院，北京 100048
中文关键词：草地贪夜蛾；时空表达；听器发育；RNA干扰
英文关键词：Spodoptera frugiperda; the temporal and spatial expression; the development of auditory organ; RNAi
中文摘要：
【目的】 研究草地贪夜蛾 Spodoptera frugiperda 听觉基因Cut和Spalt的时空表达特性及在其听器发育过程中的生物学功能，为草地贪夜蛾的害虫防治提供科学依据。【方法】 从NCBI数据库上查找并下载草地贪夜蛾Cut和Spalt基因的全长序列，并进行生物信息学分析；利用实时荧光定量PCR（Quantitative Real-time PCR，qPCR）技术检测Cut和Spalt基因在草地贪夜蛾不同发育阶段和成虫不同组织中的相对表达量；通过RT-PCR技术克隆Cut和Spalt基因的干扰片段，然后采用RNAi和qPCR技术验证干扰效率并观察虫体的发育表型。【结果】 草地贪夜蛾Cut基因的氨基酸序列与斜纹夜蛾Spodoptera litura序列相似度最高，为99.19%，Spalt基因的氨基酸序列与美洲棉铃虫Helicoverpa zea序列相似度最高，为97.15%。Cut和Spalt基因在草地贪夜蛾各发育阶段和成虫各组织中均有表达，在蛹期第7天和成虫头部的表达量最高。本研究设计并扩增获得草地贪夜蛾的Cut和Spalt基因的干扰片段，长度分别为486 bp和317 bp。RNAi表明，在幼虫第5龄期干扰可显著降低Cut和Spalt基因的相对表达量，在成虫出现听器和翅发育异常的现象。【结论】 Cut和Spalt基因的沉默会使草地贪夜蛾幼虫体内基因表达水平下调并影响成虫听觉器官和翅的发育。
英文摘要：
[Objectives] To investigate the spatio-temporal expression and biological function of the Cut and Spalt genes in Spodoptera frugiperda during the development of the auditory organ. [Methods] Full-length sequences of the Cut and Spalt genes were downloaded from the NCBI website and bioinformatics analysis performed. Quantitative Real-time PCR was used to detect the relative expression level of Cut and Spalt in different developmental stages and adult tissues. Interference fragments of Cut and Spalt were cloned with RT-PCR after which RNAi and qPCR were used to verify their effectiveness and observe the developmental phenotype of the resultant genetically modified insects. [Results] The amino acid sequence of the S. frugiperda Cut gene is most similar to that of Spodoptera litura (99.19%), whereas the amino acid sequence of the S. frugiperda Spalt gene is most similar to that of Helicoverpa zea (97.15%). Cut and Spalt were expressed in all developmental stages and adult tissues; expression was highest on the 7th day of the pupal stage and in the heads of adults. Interference fragments for S. frugiperda Cut and Spalt, with lengths of 486 bp and 317 bp, respectively, were successfully designed and amplified. RNAi significantly reduced the relative expression of Cut and Spalt in 5th instar larvae, resulting in abnormal development of the auditory organ and wing in adults. [Conclusion] Silencing Cut and Spalt down-regulated gene expression in larvae, thereby impairing normal development of the auditory organ and wing in adult S. frugiperda.