Differentially expressed microRNAs in diapausing versus HCl-treated Bombyx embryos.

Wentao Fan,Mingyue Qin,Wenchu Li,Jianrong Lin,Bimin Lin,Yangsheng Zhong,Huichao Yan,Fangyan Chen,Yun Zheng

doi:10.1371/journal.pone.0180085

Abstract

Differentially expressed microRNAs were detected to explore the molecular mechanisms of diapause termination. The total small RNA of diapause-destined silkworm eggs and HCl-treated eggs was extracted and then sequenced using HiSeq high-throughput method. 44 novel miRNAs were discovered. Compared to those in the diapause-destined eggs, 61 miRNAs showed significant changes in the acid-treated eggs, with 23 being up-regulated and 38 being down-regulated. The potential target genes of differentially expressed miRNAs were predicted by miRanda. Gene Ontology and KEGG pathway enrichment analysis of these potential target genes revealed that they were mainly located within cells and organelles, involved in cellular and metabolic processes, and participated in protein production, processing and transportation. Two differentially expressed genes, Bombyx mori SDH and Bmo-miR-2761-3p, were further analyzed with qRT-PCR. BmSDH was significantly up-regulated in the HCl-treated eggs, while Bmo-miR-2761-3p was down-regulated. These results suggested that these two genes were well coordinated in silkworm eggs. Dual luciferase reporter assay demonstrated that Bmo-miR-2761-3p inhibited the expression of BmSDH.

Highlights

Diapause is an important physiological mechanism for insects to adapt to harsh environmental conditions [1]
Identification Small RNA (sRNA) of silkworm eggs sRNA was isolated from control eggs and HCl-treated eggs on day-4 post-oviposition
HCltreated eggs showed 17049696 clean reads, while 17309699 clean reads were detected in control eggs. 9594769 and 7789512 clean reads mapped to silkworm chromosomes respectively (S1 Table). sRNA lengths were in the range of 18 to 30 nt (Fig 1). 7.79% and 9.80% of total sRNA were specific sRNA in the control eggs and HCl-treated eggs, respectively (Fig 2A). 39.31% and 44.46% were unique sRNA, respectively (Fig 2B). 8.29% and 8.49% sRNAs were miRNAs for the control group and HCl-treated group, respectively (Fig 3A and 3C)

Summary

Introduction

Diapause is an important physiological mechanism for insects to adapt to harsh environmental conditions [1]. At 25 ̊C, silkworm enters diapause at its early embryo stage 24h after oviposition [2]. A significant drop in oxygen consumption and the conversion of glycogen to sorbitol and glycerol slow down division of embryonic cells and completely stops the cells in G2 stage of the cell cycle 3 days after oviposition [3]. Diapause can be prevented if diapause-destined eggs are artificially treated with HCl. Many mechanisms for silkworm diapause have been reported. Its well known that SDH is a molecular marker for Bombyx embryo development. The activity of SDH can’t be detected in diapause-destined eggs but is measurably increased in HCl-treated eggs 2 days after oviposition [5,6].

Methods

Results

Discussion

Conclusion