Xin Hu-hu, Zhang Deng-pan, Chen Rui-ting, Cai Zi-zheng, Lu Yan, Liang Shuang, Miao Yun-gen
Institute of Sericulture and Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, People's Republic of China.
Arch Insect Biochem Physiol. 2015 Oct;90(2):59-69. doi: 10.1002/arch.21244. Epub 2015 Apr 28.
Salivary gland secretion is altered in Drosophila embryos with loss of function of the sage gene. Saliva has a reduced volume and an increased electron density according to transmission electron microscopy, resulting in regions of tube dilation and constriction with intermittent tube closure. However, the precise functions of Bmsage in silkworm (Bombyx mori) are unknown, although its sequence had been deposited in SilkDB. From this, Bmsage is inferred to be a transcription factor that regulates the synthesis of silk fibroin and interacts with another silk gland-specific transcription factor, namely, silk gland factor-1. In this study, we introduced a germline mutation of Bmsage using the Cas9/sgRNA system, a genome-editing technology, resulting in deletion of Bmsage from the genome of B. mori. Of the 15 tested samples, seven displayed alterations at the target site. The mutagenesis efficiency was about 46.7% and there were no obvious off-target effects. In the screened homozygous mutants, silk glands developed poorly and the middle and posterior silk glands (MSG and PSG) were absent, which was significantly different from the wild type. The offspring of G0 mosaic silkworms had indel mutations causing 2- or 9-bp deletions at the target site, but exhibited the same abnormal silk gland structure. Mutant larvae containing different open-reading frames of Bmsage had the same silk gland phenotype. This illustrated that the mutant phenotype was due to Bmsage knockout. We conclude that Bmsage participates in embryonic development of the silk gland.
在sage基因功能丧失的果蝇胚胎中，唾液腺分泌发生改变。根据透射电子显微镜观察，唾液的体积减小且电子密度增加，导致出现管扩张和收缩区域，并伴有间歇性管闭合。然而，家蚕（Bombyx mori）中Bmsage的确切功能尚不清楚，尽管其序列已存入SilkDB。由此推断，Bmsage是一种调节丝素蛋白合成的转录因子，并与另一种丝腺特异性转录因子丝腺因子-1相互作用。在本研究中，我们使用基因组编辑技术Cas9/sgRNA系统在家蚕中引入了Bmsage的种系突变，导致Bmsage在家蚕基因组中缺失。在15个测试样本中，有7个在靶位点出现改变。诱变效率约为46.7%，且没有明显的脱靶效应。在筛选出的纯合突变体中，丝腺发育不良，中后部丝腺（MSG和PSG）缺失，这与野生型有显著差异。G0嵌合体家蚕的后代在靶位点有导致2个或9个碱基对缺失的插入缺失突变，但丝腺结构异常相同。含有不同开放阅读框的Bmsage的突变幼虫具有相同的丝腺表型。这表明突变表型是由于Bmsage基因敲除所致。我们得出结论，Bmsage参与丝腺的胚胎发育。
