靶向调控飞蝗表皮代谢相关基因的miRNA的鉴定与分析

发布时间：2018-05-15 20:28

本文选题：飞蝗 + 表皮　；参考：《昆虫学报》2017年03期

【摘要】：【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。
[Abstract]:[objective] miRNAsmicroRNAs) is a class of non-coding RNAs widely found in eukaryotes and involved in the regulation of multiple life activities of organisms. The development of insect molting includes the formation of new epidermis and the degradation of old epidermis. The purpose of this study was to identify the key genes of epidermal metabolism of migratory locust (MiRN), and to provide an experimental basis for studying the molecular regulation mechanism of epidermal development of migratory locust (Locusta migratoria). It also provides scientific basis for the development of new molecular targets and control strategies for pest control. [methods] Bioinformatics was used to predict the potential binding of key genes to epidermis metabolism of locust migratory locust. Fluorescence quantitative PCR method was used to detect epidermis. The expression trend of metabolism-related genes and miRNAs in the 1st and 5th day nymphs of migratory locust at the 2nd and 3rd instar. Immunoprecipitation and double luciferase reporting techniques were used to analyze the binding of miRNA to its target gene in vitro and in vivo. [results] Bioinformatics was used to predict UDP-N- acetyl glucosamine binding to fatty acid synthase. The miRNAs with potential binding ability of UDP-N-acetylglucosamine pyrophorylase, asparagine-linked glycosylation protein 5 (ALG5) and Sinuous genes were miRNA-276bnmiRNA-2796 miRNA-275 and miRNA-184, respectively. Fluorescence quantitative PCR analysis showed that FASA UAPG5 and Sinuous had similar expression trend in the epidermis of second and third instar nymphs of migratory locusts. The expression trend of miRNA-276b was the same as that of miRNA-276b targeting it. The other three genes were opposite to those of miRNAs. The results of immunoprecipitation in vivo showed that the AGO1 antibody could significantly enrich the ALG5 and Sinuous genes of FASA UAPN and its targeting miRNAs. In vitro double luciferase assay showed that miRNA-276bsimiRNA-2796 miRNA-275 and miRNA-184 could significantly inhibit the expression of epidermal metabolite gene FAS-UAPG5 and Sinuous. [conclusion] this study has identified the potential miRNAss that target regulate the epidermal metabolism gene FASUAPALG5 and Sinuous of locust migratory locust. To study the regulation mechanism of epidermal miRNAs on the development of migration locust molt and the discovery of new target for pest control provided important scientific basis.
【作者单位】：山西大学应用生物学研究所;山西大学生命科学学院;中国科学院动物研究所;
【基金】：国家自然科学基金项目(31472051,31672364) 山西省科技基础条件平台建设项目(2015091010)
【分类号】：S433.2

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