白纹伊蚊miRNA的分离、鉴定及表达谱的初步分析

Isolation, identification and analysis of the expression profile of miRNAs in Aedes albopictus

目的通过Northern blotting方法验证白纹伊蚊microRNA的存在并分析其不同发育阶段的表达谱。方法根据miRBase中冈比亚按蚊的已知miRNA序列设计合成6条特异的LNA反义寡核苷酸探针,并用地高辛标记。用mirVanaTM miRNA Isolation Kit分别提取白纹伊蚊卵、一二期幼虫、三四期幼虫、蛹、雄蚊、雌蚊等六个不同发育阶段的Total RNA,15%的尿素变性PAGE电泳分离小RNA,转膜、交联、杂交、检测。结果共检测到5种miRNA的表达,其中包括3种保守的miRNA,2种蚊虫特异的miRNA,其表达方式既有组成型表达又有期特异性表达,如miR-184在各个阶段均有表达,let-7在幼虫后期才开始有表达,miR-9a主要在卵及幼虫阶段表达,miR-M1只在蚊虫卵期有表达,miR-1175在除卵期之外的其他阶段有表达而miR-1174未检测到有表达。结论首次验证了白纹伊蚊中miRNA的存在,初步证明了miRNA在白纹伊蚊发育过程中的保守性和特异性,为白纹伊蚊胚胎生长、变态发育等生命进程的研究以及新的杀虫剂的研制提供了新的思路和方法。

Objective To verify the miRNA in Aedes albopictus and characterize the expression profile of several miRNAs across all the life stages of Aedes albopictus.Methods Based on the published miRNA sequences of Anopheles gambiae and Aedes aegypti,6 DIG-labeled antisense probes were synthesized.The total RNAs from Aedes albopictus in 6 developmental stages （embryo,early larvae,late larvae,pupa,male and female adults） were extracted with a mirVana miRNA isolation kit,loaded onto 15% denaturing polyacrylamide gel and hybridized with the appropriate DIG-labeled probes.Results Northern blotting detected 5 miRNAs in Aedes albopictus,of which mir-9a was mainly expressed in embryo and larva stages,let-7 in pupa and adult stages,miR-184 in all life stages,mir-M1 only in the embryos and miR-1175 in all the life stages except for embryos.The expression profiles of these miRNAs in Aedes albopictus were similar to those in D.melanogaster and An.stepheni.miR-1174 was not detected in any of the developmental stages of Aedes albopictus.Conclusion These results present the first direct experimental evidence of miRNA in Aedes albopictus.The expression profiles of the analyzed miRNAs in Aedes albopictus showed stage specificity and conservation with other mosquitoes.Further studies on the functions of these miRNAs may offer new insights in mosquito biology and may lead to novel approaches to the development of insecticides.