New insight into the mechanism underlying the silk gland biological process by knocking out fibroin heavy chain in the silkworm
New insight into the mechanism underlying the silk gland biological process by knocking out fibroin heavy chain in the silkworm
复制标题
通过敲除蚕丝蛋白重链对丝腺生物过程的机制有了新的认识
DOI：
10.1186/s12864-018-4602-4
复制
发表时间：
2018-03-26
期刊：
影响因子：
4.4
通讯作者：
Xiang, Hui
中科院分区：
文献类型：
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作者：
Cui, Yong;Zhu, Yanan;Xiang, Hui
背景探索丝蛋白突变是否以及如何导致随后的氮重新分配，以及对丝腺降解时间的影响，对于了解丝腺生物学非常重要。家蚕基因组编辑技术的快速发展和广泛应用为我们解决这些问题提供了契机。结果利用CRISPR/Cas9系统，我们成功地对Bmfib-H进行了基因组编辑。功能丧失突变导致裸蛹和薄茧突变表型。与野生型相比，突变体的后部丝腺在丝腺细胞程序性细胞死亡之前表现出明显的降解成碎片。对五龄幼虫期第四天（L5D4）丝腺进行比较转录组分析，鉴定出后丝腺（PSG）和中部丝腺（MSG）之间有1456个差异表达基因（DEG），突变体与野生型之间有1388个差异表达基因（DEG）。所有DEG的层次聚类分别表明突变丝腺中显着下调和上调的基因进化枝。下调基因在涉及癌症、DNA 复制和细胞增殖的通路中过多。有趣的是，上调的 DEG 在蛋白酶体中显着富集。通过进一步比较野生型和突变体之间的MSG和PSG转录组，我们一致观察到突变体PSG中上调的DEG在蛋白质降解活性和蛋白酶体方面富集。与此同时，我们观察到一系列与自噬有关的上调基因。由于这些蛋白质降解过程通常发生在旋转时间之后，因此结果表明这些过程在突变体中明显提前激活。结论异常fib-H蛋白的积累可能引起蛋白酶体的激活以及自噬过程，促进此类异常蛋白质和丝腺细胞的快速降解。因此，我们的研究提出了丝蛋白突变引起的蛋白质和部分细胞降解的后续过程，这可能有助于了解其对丝腺生物过程的影响，并进一步探索蚕体内氮的重新分配。
BackgroundExploring whether and how mutation of silk protein contributes to subsequent re-allocation of nitrogen, and impacts on the timing of silk gland degradation, is important to understand silk gland biology. Rapid development and wide application of genome editing approach in the silkworm provide us an opportunity to address these issues.ResultsUsing CRISPR/Cas9 system, we successfully performed genome editing ofBmfib-H. The loss-of-function mutations caused naked pupa and thin cocoon mutant phenotypes. Compared with the wild type, the posterior silk gland of mutant showed obviously degraded into fragments in advance of programmed cell death of silk gland cells. Comparative transcriptomic analyses of silk gland at the fourth day of the fifth instar larval stage(L5D4)identified 1456 differential expressed genes (DEGs) between posterior silk gland (PSG) and mid silk gland (MSG) and 1388 DEGs between the mutant and the wild type. Hierarchical clustering of all the DEGs indicated a remarkable down-regulated and an up-regulated gene clade in the mutant silk glands, respectively. Down-regulated genes were overrepresented in the pathways involved in cancer, DNA replication and cell proliferation. Intriguingly, up-regulated DEGs are significantly enriched in the proteasome. By further comparison on the transcriptome of MSG and PSG between the wild type and the mutant, we consistently observed that up-regulated DEGs in the mutant PSG were enriched in protein degrading activity and proteasome. Meantime, we observed a series of up-regulated genes involved in autophagy. Since these protein degradation processes would be normally occur after the spinning time, the results suggesting that these progresses were activated remarkably ahead of schedule in the mutant.ConclusionsAccumulation of abnormal fib-H protein might arouse the activation of proteasomes as well as autophagy process, to promote the rapid degradation of such abnormal proteins and the silk gland cells. Our study therefore proposes a subsequent process of protein and partial cellular degradation caused by mutation of silk protein, which might be helpful for understanding its impact of the silk gland biological process, and further exploration the re-allocation of nitrogen in the silkworm.