基于RNA-Seq分析Ras1^(CA)在家蚕后部丝腺过表达对细胞周期通路基因的影响  

RNA-Seq technology based transcriptomic analysis of differentially expressed genes in the cell cycle pathway of Ras1^(CA)-overexpressed and wild type posterior silk glands of Bombyx mori

作　　者：马倩[1,2] 马俐[2] 李胜[2] 李恺[1] 

机构地区：[1]华东师范大学生命科学学院,上海200241 [2]中国科学院上海生命科学研究院植物生理生态研究所,上海200032

出　　处：《应用昆虫学报》2015年第2期390-399,共10页Chinese Journal of Applied Entomology

基　　金：国家自然科学基金(31201747);上海市自然科学基金(12ZR1452500);上海生科院优秀青年人才领域前沿项目(2012KIP305)

摘　　要：【目的】本文旨在挖掘野生型家蚕Bombyx mori和Ras1CA过表达转基因家蚕后部丝腺中的转录本差异,分析和验证细胞周期通路中的差异表达基因,从而探讨Ras1CA过表达转基因家蚕提高蚕丝产量的分子机制。【方法】利用转录组学比较野生型和Ras1CA过表达转基因家蚕后部丝腺中的基因转录本差异,经实时荧光定量PCR(q RT-PCR)验证。【结果】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺组织中有2 636个差异表达基因,其中细胞周期信号通路中有42个差异表达基因,包括细胞周期依赖性激酶(CDK)、细胞周期素(Cyclin)以及转录因子等。通过q RT-PCR检测cdk1、cyclin D1、cyclin D2、cdc7、cdh1、dp-1,2等6个基因在野生型和Ras1CA过表达转基因家蚕后部丝腺组织中的相对表达量,发现转基因家蚕中的表达量均显著高于野生型(P<0.05),其中cyclin D2的表达差异极显著(P<0.01)。q RT-PCR结果与转录本差异一致,表明Ras1CA过表达后,能够促进细胞周期通路基因的表达。【结论】野生型家蚕和Ras1CA过表达转基因家蚕后部丝腺中有大量差异表达基因,且Ras1CA能够在转录水平上调控细胞周期通路,影响后部丝腺组织的细胞分裂和器官生长,从而促进蚕丝蛋白的合成。[Objectives] To further understand the molecular mechanisms underlying the overexpression of the RaslTM oncogene in the posterior silk gland （PSG） of Bombyx mori and how this improves silk yield at the transcriptional level. Transcriptomic data from differentially expressed genes （DEGs） in the cell cycle pathway of RaslCA-overexpressed and wild type PSG were analyzed and verified by quantitative real-time PCR （qRT-PCR）. [Methods] Deep RNA sequencing of silkworm PSG was carried out and KEGG pathway enrichment analysis was conducted on the DEGs. The DEGs in the cell cycle pathway was selected to be verified by qRT-PCR. [Results] RNA-Seq revealed 2 636 DEGs in RaslCa-overexpressed PSG compared to wild type PSG. There were 42 DEGs distributed in the ＂cell cycle＂ pathway, including cyclin dependent kinases （CDKs）, eyclins and transcription factors. Consistent with the transcriptomic data, qRT-PCR verification of the selected genes; cdk1, cyclinD1, cyclinD2, cdc7, cdhl, dp-1,2, indicated that all of these were significantly upregulated by RaslcA. [Conclusion] Transcriptomic analysis revealed that there are a number of DEGs in RaslCA-overexpressed and wild type PSG. RaslCA-overexpression upregulates some DEGs in the cell cycle pathway that benefit silk gland growth and fibroin synthesis. These results advance our knowledge of the molecular mechanism underlying how RaslCA-overexpression in the PSG improves fibroin production and silk production.

关 键 词：转录组学 转基因家蚕 Ras1CA过表达 细胞周期基因 

分 类 号：Q78[生物学—分子生物学]