Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-inding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant. Overall design: To analyze the differentially expressed genes related to pigmentation and development between the wildtype silkworm strain 932VR and q-lp mutant, the epidermises in 4th instar of 932VR and q-lp are used for RNA-Seq.

本研究从普通家蚕（silkworm）品系932VR中筛选获得一株新型紫色鹌鹑样（quail-like，q-lp）突变体，该突变体表皮上的色素斑点与鹌鹑色素突变体（quail, q）相似。此外，q-lp突变体幼虫活动能力低下、取食量少且生长缓慢，死亡率较高，并伴随其他发育异常表型。家蚕表皮的色素沉着由黑色素（melanin）、眼色素（ommochrome）与蝶啶（pteridine）组成，其生长发育受蜕皮激素（ecdysone）与保幼激素（juvenile hormone）调控。本研究以932VR为对照，对蜕皮期第4龄幼虫的q-lp突变体表皮开展RNA测序（RNA-Seq）分析。结果显示，q-lp突变体中共鉴定出515个差异表达基因，其中234个基因上调表达，281个基因下调表达。BLASTGO分析表明，下调表达基因主要编码蛋白结合蛋白、膜组分、氧化还原酶及蛋白水解酶；而上调表达基因则多编码表皮结构蛋白、膜组分、转运相关蛋白与蛋白结合蛋白。本研究采用定量反转录PCR（Quantitative reverse transcription PCR）验证了RNA测序数据的准确性，重点针对三类色素及几丁质（chitin）生物合成关键基因、表皮蛋白编码基因以及若干相关核受体（nuclear receptor）基因展开验证，上述基因被认为在q-lp突变体中发挥关键调控作用。基于实验结果，我们得出以下三点结论：1）q-lp突变体中黑色素、眼色素及蝶啶三类色素均显著上调；2）q-lp突变体的表皮蛋白表达量高于932VR品系，且上调表达的表皮蛋白基因数量显著多于下调基因；3）q-lp突变体中蜕皮激素调控的下游通路发生阻断。本研究结果为进一步解析q-lp突变体的发育变化机制奠定了基础。实验设计：为分析野生型家蚕品系932VR与q-lp突变体之间与色素沉着及发育相关的差异表达基因，本研究分别采集932VR与q-lp突变体第4龄幼虫的表皮组织进行RNA测序（RNA-Seq）分析。