靶向调控飞蝗表皮代谢相关基因的miRNA的鉴定与分析

Identification and analysis of the miRNAs targeting key genes involved in cuticle metabolism in Locusta migratoria (Orthoptera:Acrididae)

【目的】miRNAs(microRNAs)是一类广泛存在于真核生物中并参与调控生物体多种生命活动的非编码RNA。昆虫蜕皮发育过程包括新表皮的生成和旧表皮的降解。本研究旨在鉴定靶向调控飞蝗表皮代谢关键基因的miRNAs,为研究飞蝗Locusta migratoria表皮发育的分子调控机制提供一定的实验基础,同时为研发新的害虫防治分子靶标和防治策略提供科学依据。【方法】采用生物信息学方法预测与飞蝗表皮代谢关键基因潜在结合的miRNAs;荧光定量PCR方法检测表皮代谢相关基因及以其为靶标的miRNAs在飞蝗2龄和3龄第1,3和5天若虫中的表达趋势;利用免疫共沉淀技术及双荧光素酶报告技术在体内外水平分析miRNA与其靶基因的结合情况。【结果】生物信息学方法预测到与脂肪酸合成酶(fatty acid synthase,FAS)、UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UDP-N-acetylglucosamine pyrophorylase,UAP)、糖基转移酶(asparagine-linked glycosylation protein 5,ALG5)和Sinuous等表皮代谢相关酶基因有潜在结合能力的miRNAs分别为miRNA-276b,miRNA-2796,miRNA-275和miRNA-184。荧光定量PCR分析表明,FAS,UAP,ALG5以及Sinuous在飞蝗2龄和3龄不同日龄若虫表皮中具有相似的表达趋势,FAS与以其为靶标的miRNA-276b表达趋势相同,其余3个基因与以其为靶标的miRNAs的表达趋势相反。通过体内免疫共沉淀研究发现,AGO1抗体可显著富集FAS,UAP,ALG5和Sinuous基因以及以其为靶标的miRNAs。体外双荧光素酶实验发现,miRNA-276b,miRNA-2796,miRNA-275和miRNA-184对表皮代谢基因FAS,UAP,ALG5和Sinuous的表达有较明显的抑制作用。【结论】本研究鉴定了靶向调控飞蝗表皮代谢基因FAS,UAP,ALG5和Sinuous的潜在miRNAs,为进一步研究表皮miRNAs对飞蝗蜕皮发育的调控机制及害虫防治新靶标的发现提供了重要科学依据。

【Aim】MicroRNAs（ miRNAs） are a class of non-coding RNAs that are widely present in eukaryotes and involved in the regulation of many life processes. The formation of new cuticle and the degradation of old cuticle are necessary for insect molting. This study aims to identify the miRNAs targeting key genes involved in cuticle metabolism in the locust Locusta migratoria,which may provide an experimental foundation for investigating the molecular regulation mechanism of cuticle metabolism andalso be helpful to developing new molecular targets for pest control. 【Methods】Potential miRNAs that bind key genes involved in cuticle metabolism of L. migratoria were identified by using bioinformatics approaches. Reverse transcription quantitative PCR（ RT-q PCR） was used to detect the expression patterns of cuticular genes and the miRNAs targeting them in the day-1,day-3 and day-5 2nd and 3rd instar nymphs. The RNA immunoprecipitation（ RIP） and dual luciferase reporter assays were performed to validate the potential binding relationship of miRNAs and their target genes in vivo or in vitro.【Results】The bioinformatics prediction results showed that four cuticle metabolism related genes from L.migratoria,i. e.,fatty acid synthase（ FAS） gene,UDP-N-acetylglucosamine pyrophosphorylase（ UAP）gene,asparagine-linked glycosylation protein 5（ ALG5） gene and Sinuous gene,contain potential binding sites for miRNA-276 b,miRNA-2796,miRNA-275 and miRNA-184,respectively. RT-q PCR analysis showed that FAS,UAP,ALG5 and Sinuous exhibited similar expression patterns in the cuticle of different day-old 2nd and 3rd instar nymphs. The expression pattern of FAS presented a positive correlation with that of miRNA-276 b targeting it,whereas the expression patterns of the other three genes were negatively correlated with those of the miRNAs targeting them respectively. RIP assays suggested that FAS,UAP,ALG5 and Sinuous,as well as the miRNAs targeting them,were significantly enriched in vivo. Dual luciferase reporter assay showed that miRNA-276 b,miRNA-2796,miRNA-275 and miRNA-184 had a significant inhibitory effect on the expressions of FAS,UAP,ALG5 and Sinuous genes,respectively,in vitro. 【Conclusion 】 We identified the miRNAs targeting four cuticle genes from L.migratoria,which provides the basis for further study of the regulation mechanism of miRNAs on molt development and the discovery of new targets for pest control.