Guangzhou Key Laboratory of Insect Development Regulation and Application Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China.
Center for Ecological and Environmental Sciences, Northwestern Polytechnical University, Xi'an, 710129, China.
BMC Genomics. 2018 Mar 26;19(1):215. doi: 10.1186/s12864-018-4602-4.
Exploring 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.
Using CRISPR/Cas9 system, we successfully performed genome editing of Bmfib-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.
Accumulation 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.
探索丝蛋白突变是否以及如何导致氮的重新分配，并影响丝腺降解的时间，对于理解丝腺生物学是很重要的。基因组编辑方法在蚕中的快速发展和广泛应用为我们提供了一个解决这些问题的机会。
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，突变体的后丝腺在丝腺细胞程序性死亡之前明显提前降解成碎片。第五龄幼虫期第四天（L5D4）后丝腺（PSG）和中丝腺（MSG）之间的比较转录组分析鉴定出 1456 个差异表达基因（DEGs），突变体和野生型之间有 1388 个 DEGs。所有 DEGs 的层次聚类表明，突变体丝腺中存在一个明显下调和一个上调的基因簇。下调的基因在涉及癌症、DNA 复制和细胞增殖的途径中过表达。有趣的是，上调的 DEGs 显著富集在蛋白酶体中。通过进一步比较野生型和突变体 MSG 和 PSG 的转录组，我们一致观察到突变体 PSG 中上调的 DEGs 富集在蛋白降解活性和蛋白酶体中。同时，我们观察到一系列参与自噬的上调基因。由于这些蛋白降解过程通常发生在吐丝时间之后，结果表明这些过程在突变体中提前显著激活。
异常纤维蛋白 H 蛋白的积累可能会引起蛋白酶体以及自噬过程的激活，从而促进异常蛋白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后引起的蛋白质和部分细胞降解的后续过程，这有助于理解其对丝腺生物学过程的影响，并进一步探索蚕中氮的再分配。
