Research Article: De novo characterization of microRNAs in oriental fruit moth Grapholita molesta and selection of reference genes for normalization of microRNA expression

Date Published: February 3, 2017

Publisher: Public Library of Science

Author(s): Xiu Wang, Yisong Li, Jing Zhang, Qingwen Zhang, Xiaoxia Liu, Zhen Li, Youjun Zhang.


MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that have critical regulatory functions in almost all known biological processes at the post-transcriptional level in a variety of organisms. The oriental fruit moth Grapholita molesta is one of the most serious pests in orchards worldwide and threatens the production of Rosacea fruits. In this study, a de novo small RNA library constructed from mixed stages of G. molesta was sequenced through Illumina sequencing platform and a total of 536 mature miRNAs consisting of 291 conserved and 245 novel miRNAs were identified. Most of the conserved and novel miRNAs were detected with moderate abundance. The miRNAs in the same cluster normally showed correlated expressional profiles. A comparative analysis of the 79 conserved miRNA families within 31 arthropod species indicated that these miRNA families were more conserved among insects and within orders of closer phylogenetic relationships. The KEGG pathway analysis and network prediction of target genes indicated that the complex composed of miRNAs, clock genes and developmental regulation genes may play vital roles to regulate the developmental circadian rhythm of G. molesta. Furthermore, based on the sRNA library of G. molesta, suitable reference genes were selected and validated for study of miRNA transcriptional profile in G. molesta under two biotic and six abiotic experimental conditions. This study systematically documented the miRNA profile in G. molesta, which could lay a foundation for further understanding of the regulatory roles of miRNAs in the development and metabolism in this pest and might also suggest clues to the development of genetic-based techniques for agricultural pest control.

Partial Text

MicroRNAs (miRNAs) are non-coding small RNAs and are usually 21–24 nucleotide (nt) in lengths [1,2]. Accumulative reports suggest that miRNAs function as important gene expression regulators in almost all known physiological and pathophysiological process in varieties of eukaryotes and viruses [3–5]. Through the specific recognition between the miRNA seed region (nucleotides 2–8 from the 5’ en≈d) and the target sequences of mRNA existing in the 3’, 5’–untranslated region (3’UTR, 5’UTR) or the open reading frame, the finely tuning results of miRNAs normally lead to the degradation [6], transcriptional or translational repression [7,8] or even transcriptional enhancement of the target mRNA [9,10].

The miRNA information of several Lepidopteran insects has been identified with reference to their genomic information [20,61] or genome sequences of the model insect silkworm (B. mori) [62,63]. In the present study, a pooled sRNA library of G. molesta was prepared from mixed developmental stages of oriental fruit moth, and was further analyzed in reference to its own transcriptome and the genome of D. plexippus which was found with the highest similarity to the transcripts of G. molesta in the phylogenetic conservation analysis (unpublished data). A total of 536 mature miRNAs composed of 291 previously reported and 245 novel ones were finally identified in G. molesta. In previous studies of Lepidopteran insects, 55 conserved and 202 novel miRNAs were found in B. mori [61], 163 conserved and 13 novel miRNAs were identified in M. sexta [20], and 97, 91 and 69 conserved together with 1, 8 and 383 novel ones were respectively identified in the miRNA analyses of Helicoverpa armigera, Spodoptera litura and P. xylostella [62,63]. With the cumulative availability of miRNA data and the increase of sequencing depth, more information of mature miRNA would be uncovered and more novel miRNA could be identified in lepidopterans, which would provide useful repertoires for clarification of the modulation complex in insects and other organisms.