CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells.
CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells.
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CRISPR介导的丝素重链基因的内源性激活触发家蚕胚胎细胞的细胞应激反应。
DOI：
10.3390/insects12060552
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发表时间：
2021-06-13
期刊：
影响因子：
3
通讯作者：
Xia Q
中科院分区：
文献类型：
--
作者：
Hu W;Wang X;Ma S;Peng Z;Cao Y;Xia Q
基于CRISPRa方法，在家蚕胚胎（BmE）细胞中进行激活内源性丝素重链（FibH）基因的表达，其由dCas 9-VPR（由VP 64、p65和Rta组成的三联激活剂）和靶向FibH基因启动子的sgRNA的组合驱动，以研究FibH在丝腺细胞发育中的生物学作用。内源性FibH基因的激活导致细胞应激反应相关基因的上调，提示激活的FibH基因表达与细胞应激反应之间存在显著正相关。本研究结果为研究丝素分泌障碍引起的细胞应激反应提供了一个潜在的模型，并为深入了解丝腺的发育过程奠定了基础。家蚕是一种重要的经济昆虫，因为它是丝绸的主要生产者。丝素重链基因（FibH）编码丝蛋白的核心成分，在丝腺细胞中特异性高表达，而在其它细胞中不表达。虽然家蚕FibH基因在转录调控方面已经得到了很好的研究，但其在丝腺细胞发育中的生物学功能仍不清楚。在本研究中，我们构建了一个CRISPRa系统来激活家蚕胚胎（BmE）细胞中FibH的内源性转录，并且成功激活了FibH的mRNA表达。此外，我们发现在以9：1的摩尔比瞬时转染sgRNA/dCas 9-VPR后60小时FibH表达增加至最大值。qRT-PCR分析显示，随着FibH基因的激活，细胞应激反应相关基因的表达水平均显著上调，上调幅度沿着。此外，溶血示踪剂红和单丹酰尸胺（MDC）染色试验显示，在FibH激活的BmE细胞的自噬明显的外观。因此，我们认为FibH基因的激活导致BmE细胞中细胞应激反应相关基因的上调，这对于理解B的丝腺发育和丝素蛋白分泌过程是必要的。森。
Based on a CRISPRa approach, activating endogenous fibroin heavy chain (FibH) gene expression in Bombyx mori embryonic (BmE) cells, which was driven by a combination of the dCas9-VPR (a tripartite activator, composed of VP64, p65, and Rta) and the sgRNA targeting to the promoter of FibH gene, was performed for investigating the biological roles of FibH in the development of silk gland cells. The activation of the endogenous FibH gene lead to up-regulation of cellular stress responses-related genes, which suggested a significant positive correlation between activated FibH gene expression and cellular stress responses. Moreover, the present findings might provide a potential model for studying the cellular stress responses caused by silk secretion disorder and lay a foundation for the understanding of silk gland development in silk-spinning insects. The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.
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