基于RNA干扰的家蝇几丁质酶2的幼虫转录组测序  

Transcriptome analysis of Musca domestica larvae with RNAi of chitinase 2 gene

作　　者：苏佩佩 国果 赵欣宇[1] 杨隆兵 朱丽娟 田竺青 朱贵明 尚小丽 SU Peipei;GUO Guo;ZHAO Xinyu;YANG Longbing;ZHU Lijuan;TIAN Zhuqing;ZHU Guiming;SHANG Xiaoli(Key and Characteristic Laboratory of Modern Pathogen Biology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,China;Key Laboratory of Environmental Pollution Monitoring and Disease Control of Ministry of Education,Guizhou Medical University,Guiyang 550025,China;School of Food Science,Guizhou Medical University,Guiyang 550025,China)

机构地区：[1]贵州医科大学基础医学院现代病原生物学重点特色实验室,贵阳550025 [2]贵州医科大学环境污染与疾病监控教育部重点实验室,贵阳550025 [3]贵州医科大学食品科学学院,贵阳550025

出　　处：《应用与环境生物学报》2021年第1期177-184,共8页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(81560337,81760647,31560253);贵州省教育厅创新群体重大研究项目(黔教合KY字[2016]031)资助。

摘　　要：几丁质酶是昆虫几丁质降解过程中的重要酶类,在昆虫整个生长发育过程中发挥着重要作用.为筛选及挖掘与防治有害医学昆虫相关的分子靶标,根据家蝇几丁质酶2(Musca domestica chitinase 2,MdCht2)基因序列设计并合成双链RNA(double-stranded RNA,dsRNA),采用微量注射法向家蝇2龄幼虫导入dsRNA,对对照组及干扰组的样本进行转录组测序,筛选差异表达基因,进行GO(gene ontology)功能注释和KEGG(kyoto encyclopedia of genes and genomes)通路富集分析.结果显示,注射dsRNA 24 h后,幼虫MdCht2 mRNA的表达显著下降,提示导入的dsRNA干扰了MdCht2的表达.经转录组测序,与对照组相比,显著差异基因共213条,其中78条基因为上调表达,135条基因为下调表达.随机选取8条显著差异基因通过实时荧光定量PCR(qRT-PCR)进行验证,该结果与转录组结果趋势一致.根据功能注释发现这些差异基因与生长发育、脂类代谢、免疫调控等途径相关.本研究通过RNA干扰和转录组测序技术处理家蝇获得大量差异基因,结果可为后续几丁质酶相关基因的挖掘和鉴定及功能研究提供一定的数据基础.(图7表4参46)Chitinase is an important enzyme in the degradation of insect chitin and plays essential roles in the growth and development of insects. In this study, double-stranded RNA(dsRNA) targeting the chitinase 2(MdCht2) gene of Musca domestica was injected into the 2 nd instar larvae of M. domestica to determine the interference with the expression of the MdCht2 gene. The transcriptomes of the control and MdCht2 RNAi larvae were sequenced, and differentially expressed genes were screened for GO(Gene Ontology) functional annotation and KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. At 24 h post dsRNA injection, the expression of MdCht2 in larvae decreased significantly, suggesting that the introduced dsRNA interfered with the expression of MdCht2. Using transcriptome sequencing, differential transcription of 213 genes(78 up-and 135 down-regulated genes) in MdCht2 RNAi larvae was observed compared with the control group. Eight significant differentially expressed genes were randomly selected and verified by qRT-PCR. The results were consistent with the transcriptomic results, confirming that the transcriptomic data were reliable. According to the functional annotation, these differentially expressed genes were involved in growth and development, lipid metabolism, immune regulation, and other pathways, indicating that MdCht2 is an important gene affecting the development of M. domestica.

关 键 词：家蝇 MdCht2 RNA干扰 转录组测序 

分 类 号：Q963[生物学—昆虫学]