College of Animal Sciences, Zhejiang University, Hangzhou, China.
PLoS One. 2013 Aug 2;8(8):e70118. doi: 10.1371/journal.pone.0070118. Print 2013.
Reactive oxygen species (ROS) from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and their related dual oxidases are known to have significant roles in innate immunity and cell proliferation. In this study, the 5,545 bp cDNA of the silkworm Bombyx mori dual oxidase (BmDuox) gene containing a full-length open reading frame was cloned. It was shown to include an N-terminal signal peptide consisting of 28 amino acid residues, a 240 bp 5'-terminal untranslated region (5'-UTR), an 802 bp 3'-terminal region (3'-UTR), which contains nine ATTTA motifs, and a 4,503 bp open reading frame encoding a polypeptide of 1,500 amino acid residues. Structural analysis indicated that BmDuox contains a typical peroxidase domain at the N-terminus followed by a calcium-binding domain, a ferric-reducing domain, six transmembrane regions and binding domains for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). Transcriptional analysis revealed that BmDuox mRNA was expressed more highly in the head, testis and trachea compared to the midgut, hemocyte, Malpighian tube, ovary, fat bodies and silk glands. BmDuox mRNA was expressed during all the developmental stages of the silkworm. Subcellular localization revealed that BmDoux was present mainly in the periphery of the cells. Some cytoplasmic staining was detected, with rare signals in the nucleus. Expression of BmDuox was induced significantly in the larval midgut upon challenge by Escherichia coli and Bombyx mori nucleopolyhedrovirus (BmNPV). BmDuox-deleted larvae showed a marked increase in microbial proliferation in the midgut after ingestion of fluorescence-labeled bacteria compared to the control. We conclude that reducing BmDuox expression greatly increased the bacterial load, suggesting BmDuox has an important role in inhibiting microbial proliferation and the maintenance of homeostasis in the silkworm midgut.
活性氧（ROS）来自烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶及其相关的双氧化酶，已知它们在先天免疫和细胞增殖中具有重要作用。在这项研究中，克隆了含有全长开放阅读框的家蚕双氧化酶（BmDuox）基因的 5545bp cDNA。结果表明，它包含一个由 28 个氨基酸残基组成的 N 端信号肽，一个 240bp 的 5'-非翻译区（5'-UTR），一个 802bp 的 3'-末端区（3'-UTR），其中包含 9 个 ATTTA 基序，以及一个 4503bp 的开放阅读框，编码一个由 1500 个氨基酸残基组成的多肽。结构分析表明，BmDuox 在 N 端包含一个典型的过氧化物酶结构域，其后是一个钙结合结构域、一个还原型铁结合结构域、六个跨膜区以及黄素腺嘌呤二核苷酸（FAD）和烟酰胺腺嘌呤二核苷酸（NAD）的结合结构域。转录分析显示，与中肠、血细胞、马氏管、卵巢、脂肪体和丝腺相比，BmDuox mRNA 在头部、睾丸和气管中表达水平更高。BmDuox mRNA 在蚕的所有发育阶段都有表达。亚细胞定位显示 BmDoux 主要存在于细胞的外周。细胞质中检测到一些染色，核内信号很少。当幼虫中肠受到大肠杆菌和家蚕核型多角体病毒（BmNPV）的挑战时，BmDuox 的表达显著诱导。与对照相比，摄入荧光标记细菌后，BmDuox 缺失的幼虫中肠微生物增殖明显增加。我们得出结论，降低 BmDuox 的表达大大增加了细菌负荷，表明 BmDuox 在抑制微生物增殖和维持家蚕中肠内稳态方面具有重要作用。
