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当前位置: 首页 > 文章 > miRNA调控昆虫精巢干细胞自我更新相关基因的保守性分析和靶标验证 昆虫学报 2017 (8) 847-856
Position: Home > Articles > Function validation and evolution analysis of miRNA regulation of insect testis stem cell self-renewal associated genes Acta Entomologica Sinica 2017 (8) 847-856

miRNA调控昆虫精巢干细胞自我更新相关基因的保守性分析和靶标验证

作  者：
马行洲;李飞
单  位：
浙江大学昆虫科学研究所;南京农业大学植物保护学院
关键词：
昆虫;家蚕;小菜蛾;精巢;干细胞;自我更新;miRNA靶标;3'UTR
摘  要：
【目的】发现昆虫精巢干细胞自我更新相关基因3'UTR上的保守miRNA靶位点,为深入研究昆虫精子发生进程中的miRNA-基因调控关系提供参考。【方法】使用Blast搜索27种全变态昆虫精巢干细胞自我更新相关基因,并预测这些基因的3'UTR。使用Target Scan预测这些基因3'UTR上的miRNA靶位点。对家蚕Bombyx mori和小菜蛾Plutella xylostella的Chinmo和Imp基因包含let-7-5p靶标点的3'UTR序列进行了克隆,使用双荧光素酶报告系统对其靶标关系进行验证。【结果】在27种全变态昆虫中发现了17个精巢干细胞自我更新相关基因。利用Target Scan在这些基因的3'UTR上预测到了203个保守的miRNA靶位点。其中Zfh-1,Chinmo,Ken及Imp的3'UTR上存在大量的miRNA靶位点,但miRNA靶位点数目在不同昆虫类群中也存在很大差异。其中,Chinmo及Imp 3'UTR上的let-7-5p靶位点在昆虫中保守。分别克隆了家蚕和小菜蛾Chinmo和Imp的3'UTR,通过双荧光素酶报告系统转染239T细胞证实了let-7-5p对Chinmo和Imp两个基因的调控作用。【结论】发现在4个昆虫精巢干细胞自我更新相关基因Zfh-1,Chinmo,Ken和Imp的3'UTR上存在大量的miRNA靶标位点。其中Chinmo及Imp基因3'UTR上let-7-5p靶位点在大多数全变态昆虫中保守,双荧光素酶报告系统实验证实了家蚕和小菜蛾中let-7-5p与Chinmo和Imp存在相互作用,为进一步研究miRNA在昆虫精子发生进程中的功能提供了参考。
译  名：
Function validation and evolution analysis of miRNA regulation of insect testis stem cell self-renewal associated genes
作  者：
MA Xing-Zhou;LI Fei;College of Plant Protection,Nanjing Agricultural University;Institute of Insect Science,Zhejiang University;
关键词：
Insect;;Bombyx mori;;Plutella xylostella testis;;stem cell;;self-renewal;;miRNA target site;;3'UTR
摘  要：
【Aim】To search conserved miRNA target sites on the 3'UTRs of insect testis stem cell selfrenewal associated genes,and to provide references for studying the miRNA-gene relationship in the process of insect spermatogenesis. 【Methods 】 Testis stem cell self-renewal associated genes were identified in 27 holometabolan insects by BLAST search,and the 3' UTRs of these genes were predicted.The miRNA target sites on the 3'UTRs of these genes were predicted by using Target Scan. The 3'UTRs of Chinmo and Imp containing let-7-5p target site of Bombyx mori and Plutella xylostella were cloned,and the target-gene relationship was verified by dual-luciferase reporter system. 【Results】Seventeen testis stem cell self-renewal associated genes were identified in 27 holometabolan insects. By using Target Scan,203 miRNA target sites were identified. A high number of conserved miRNA target sites were found on the 3'UTRs of Zfh-1,Chinmo,Ken and Imp. The number of conserved miRNA target sites was different among various insects. The let-7-5p target sites on Chinmo and Imp were conserved in insects and could be confirmed by other miRNA target prediction programs. The 3' UTRs of these two genes were clonedfrom B. mori and P. xylostella. The dual-luciferase reporter system in 293 T cells validated the regulation of let-7-5p on Chinmo and Imp from these two insect species. 【Conclusion】In this study,a large number of miRNA target sites were found on the 3' UTRs of Zfh-1,Chinmo,Ken and Imp. The let-7-5p target sites on 3' UTRs of Chinmo and Imp are conserved in holometabolan insects. The target relationships between let-7-5p and Chinmo or Imp genes of B. mori and P. xylostella were verified by dual luciferase reporter system. This study will be helpful for better understanding the functions of miRNA in insect spermatogenesis.

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