Comparative transcriptomes analysis of the wing disc between two silkworm strains with different size of wings

Wings of Bombyx mori (B. mori) develop from the primordium, and different B. mori strains have different wing types. In order to identify the key factors influencing B. mori wing development, we chose strains P50 and U11, which are typical for normal wing and minute wing phenotypes, respectively. We dissected the wing disc on the 1st-day of wandering stage (P50D1 and U11D1), 2nd-day of wandering stage (P50D2 and U11D2), and 3rd-day of wandering stage (P50D3 and U11D3). Subsequently, RNA-sequencing (RNA-Seq) was performed on both strains in order to construct their gene expression profiles. P50 exhibited 628 genes differentially expressed to U11, 324 up-regulated genes, and 304 down-regulated genes. Five enriched gene ontology (GO) terms were identified by GO enrichment analysis based on these differentially expressed genes (DEGs). KEGG enrichment analysis results showed that the DEGs were enriched in five pathways; of these, we identified three pathways related to the development of wings. The three pathways include amino sugar and nucleotide sugar metabolism pathway, proteasome signaling pathway, and the Hippo signaling pathway. The representative genes in the enrichment pathways were further verified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The RNA-Seq and qRT-PCR results were largely consistent with each other. Our results also revealed that the significantly different genes obtained in our study might be involved in the development of the size of B. mori wings. In addition, several KEGG enriched pathways might be involved in the regulation of the pathways of wing formation. These results provide a basis for further research of wing development in B. mori.

家蚕（Bombyx mori, B. mori）的翅膀由翅原基发育而成，不同家蚕品系具有差异化的翅型。为明确影响家蚕翅膀发育的关键调控因子，本研究选取分别以正常翅、微小翅为典型表型的P50与U11两个品系作为实验材料。分别于徘徊期第1天（P50D1、U11D1）、第2天（P50D2、U11D2）及第3天（P50D3、U11D3）采集翅盘组织进行解剖。随后对两个品系开展RNA测序（RNA-sequencing, RNA-Seq），以构建其基因表达谱。相较于U11品系，P50品系中共鉴定出628个差异表达基因（differentially expressed genes, DEGs），其中324个基因表达上调，304个基因表达下调。基于上述差异表达基因进行基因本体（Gene Ontology, GO）富集分析，共鉴定出5个显著富集的GO条目。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析结果显示，差异表达基因富集于5条信号通路，其中3条与家蚕翅膀发育密切相关，分别为氨基糖与核苷酸糖代谢通路、蛋白酶体信号通路以及Hippo信号通路。进一步通过实时定量反转录聚合酶链反应（quantitative real-time reverse transcription polymerase chain reaction, qRT-PCR）对富集通路中的代表性基因进行验证，RNA-seq与qRT-PCR的检测结果总体一致性良好。本研究结果同时表明，筛选得到的显著差异表达基因可能参与家蚕翅膀尺寸的发育调控；此外，若干KEGG富集通路可能参与翅膀形成相关通路的调控过程。上述研究结果为家蚕翅膀发育的后续深入研究提供了重要的理论基础。