›› 2017, Vol. 60 ›› Issue (3): 309-317.doi: 10.16380/j.kcxb.2017.03.008

• RESEARCH PAPERS • Previous Articles     Next Articles

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

WANG Yan-Li1,2, YANG Mei-Ling3, MA En-Bo1, ZHANG Jian-Zhen1,*   

  1. (1. Institute of Applied Biology, Shanxi University, Taiyuan 030006, China; 2. College of Life Sciences, Shanxi University, Taiyuan 030006, China; 3. Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China)
  • Online:2017-03-20 Published:2017-03-20

Abstract: 【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 and also 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-qPCR) 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-276b, miRNA-2796, miRNA-275 and miRNA-184, respectively. RT-qPCR 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-276b 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-276b, 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.

Key words: Locusta migratoria, cuticle, miRNA, target gene, molt development, gene expression