miR-252-5p通过靶向调控保幼激素酸甲基转移酶基因JHAMT的表达而影响果蝇变态发育

doi:10.16380/j.kcxb.2022.07.002

摘要/Abstract

摘要： 【目的】保幼激素酸甲基转移酶(juvenile hormone acid methyl transferase, JHAMT)是保幼激素(juvenile hormone, JH)合成通路中的关键限速酶。本研究旨在筛选并验证靶向调控黑腹果蝇Drosophila melanogaster JHAMT转录表达的miRNA，揭示miRNA在JH生物合成中的作用机理。【方法】首先通过miRanda, TargetScan和microT-CDS在线网站对靶向黑腹果蝇JHAMT的miRNA进行预测，取3个网站均能预测到的miRNA作为候选靶向JHAMT的miRNA；利用双荧光素酶系统对候选miRNA与JHAMT的靶向关系进行验证；qRT-PCR检测候选miRNA与JHAMT在黑腹果蝇生长发育中的表达模式；利用qRT-PCR和果蝇GAL4-UAS系统分别检测黑腹果蝇咽侧体中超表达miRNA对JHAMT的表达以及对黑腹果蝇变态发育的影响。【结果】 miRanda, TargetScan和microT-CDS分别预测到5, 18和16个靶向JHAMT的miRNA，共同预测到4个miRNA，分别是miR-252-5p, miR-277-3p, miR-1002-5p和miR-987-5p。双荧光素酶检测结果表明，miR-252-5p mimics可显著降低野生型JHAMT 3′UTR荧光素酶报告基因载体所表达的荧光素酶活性，而JHAMT 3′UTR区中miR-252-5p结合位点突变后，该抑制作用被解除。qRT-PCR检测结果表明，miR-252-5p与JHAMT在黑腹果蝇卵、幼虫及预蛹期的转录表达模式相反。咽侧体中超表达miR-252后，可显著降低JHAMT和JH初级反应基因Kr-h1的表达水平；且表现出类似JH缺失的表型，如化蛹时间推迟、体重变轻以及蛹期死亡增加。【结论】miR-252-5p可通过靶向作用于JHAMT参与JH生物合成调控，从而影响果蝇变态发育。

关键词: 黑腹果蝇, miRNA, 靶基因, 保幼激素甲基转移酶, miR-252-5p, 变态发育

Abstract: 【Aim】 Juvenile hormone acid methyl transferase (JHAMT) is the key rate-limiting enzyme in the juvenile hormone (JH) synthetic pathway. This study aims to screen and verify the miRNAs targetedly regulating the transcription of JHAMT, and to eveal the important action mechanism of miRNAs in JH biosynthesis in Drosophila melanogaster. 【Methods】 MiRNAs targeting JHAMT in D. melanogaster were predicted using online websites miRanda, TargetScan and microT-CDS, and those predicted by all the three websites were selected as the miRNA candidates targeting JHAMT. The targeted relationship between candidate miRNAs and JHAMT were verified using a dual luciferase assay system. The expression profiles of miRNA and JHAMT during D. melanogaster development were detected by qRT-PCR. The effects of miRNAs on the JHAMT expression and metamorphosis in D. melanogaster were tested by overexpressing miRNA in the corpora allata using qRT-PCR and the fly GAL4-UAS system, respectively. 【Results】 A total of 5, 18 and 16 miRNAs targeting JHAMT were predicted by miRanda, TargetScan and microT-CDS, respectively, and four miRNAs including miR-252-5p, miR-277-3p, miR-1002-5p and miR-987-5p were coincidentally predicted by these three algorithms. Dual luciferase assay results showed that miR-252-5p mimics significantly decreased the luciferase activities of wild-type JHAMT 3′UTR luciferase reporter, and the suppression effect was compromised when the miR-252-5p binding sites within the 3′UTR of JHAMT were mutated. qRT-PCR results showed that miR-252-5p and JHAMT displayed opposite expression patterns in egg, larval and prepupal stages of D. melanogaster, and overexpression of miR-252 in the corpora allata significantly decreased the expression levels of JHAMT and the JH primary response gene Kr-h1. Meanwhile, overexpression of miR-252 resulted in similar phenotypes as JH deletion, such as delayed pupation, reduced pupal size and increased pupal lethality. 【Conclusion】 miR-252-5p affects Drosophila metamorphosis through targeting JHAMT and regulating JH biosynthesis.

Key words: Drosophila melanogaster, miRNA, target gene, juvenile hormone acid methyl transferase, miR-252-5p, metamorphosis

张泽麟, 嵇杰, 徐文雨, 刘卓, 范晓春, 邓昌鑫, 何倩毓. miR-252-5p通过靶向调控保幼激素酸甲基转移酶基因JHAMT的表达而影响果蝇变态发育[J]. 昆虫学报, 2022, 65(7): 799-806.

ZHANG Ze-Lin, JI Jie, XU Wen-Yu, LIU Zhuo, FAN Xiao-Chun, DENG Chang-Xin, HE Qian-Yu. miR-252-5p affects metamorphosis by targetedly regulating the expression of juvenile hormone acid methyl transferase gene JHAMTin Drosophila[J]. Acta Entomologica Sinica, 2022, 65(7): 799-806.

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miR-252-5p通过靶向调控保幼激素酸甲基转移酶基因JHAMT的表达而影响果蝇变态发育

miR-252-5p affects metamorphosis by targetedly regulating the expression of juvenile hormone acid methyl transferase gene JHAMTin Drosophila

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