Department of Bioinformatics and Molecular Neuropathology, Meiji Pharmaceutical University, Tokyo, Japan.
PLoS One. 2011 Mar 24;6(3):e17683. doi: 10.1371/journal.pone.0017683.
We cloned cDNA for the Bombyx mori DJ-1 protein (BmDJ-1) from the brains of larvae. BmDJ-1 is composed of 190 amino acids and encoded by 672 nucleotides. Northern blot analysis showed that BmDJ-1 is transcribed as a 756-bp mRNA and has one isoform. Reverse transcriptase (RT)-PCR experiments revealed that the BmDJ-1 was present in the brain, fatbody, Malpighian tubule, ovary and testis but present in only low amounts in the silkgland and hemocyte of day 4 fifth instar larvae. Immunological analysis demonstrated the presence of BmDJ-1 in the brain, midgut, fatbody, Malpighian tubule, testis and ovary from the larvae to the adult. We found that BmDJ-1 has a unique expression pattern through the fifth instar larval to adult developmental stage. We assessed the anti-oxidative function of BmDJ-1 using rotenone (ROT) in day 3 fifth instar larvae. Administration of ROT to day 3 fifth instar larvae, together with exogenous (BmNPV-BmDJ-1 infection for 4 days in advance) BmDJ-1, produced significantly lower 24-h mortality in BmDJ-1 groups than in the control. 2D-PAGE revealed an isoelectric point (pI) shift to an acidic form for BmDJ-1 in BmN4 cells upon ROT stimulus. Among the factors examined for their effects on expression level of BmDJ-1 in the hemolymph, nitric oxide (NO) concentration was identified based on dramatic developmental stage-dependent changes. Administration of isosorbide dinitrate (ISDN), which is an NO donor, to BmN4 cells produced increased expression of BmDJ-1 compared to the control. These results suggest that BmDJ-1 might control oxidative stress in the cell due to NO and serves as a development modulation factor in B. mori.
我们从幼虫脑中克隆了家蚕 DJ-1 蛋白（BmDJ-1）的 cDNA。BmDJ-1 由 190 个氨基酸组成，由 672 个核苷酸编码。Northern blot 分析表明，BmDJ-1 转录为 756bp 的 mRNA，有一个同工型。逆转录（RT）-PCR 实验表明，BmDJ-1 存在于脑中、脂肪体、马氏管、卵巢和睾丸中，但在第 4 天 5 龄幼虫的丝腺和血细胞中含量较低。免疫分析表明，BmDJ-1 存在于幼虫至成虫的脑中、中肠、脂肪体、马氏管、睾丸和卵巢中。我们发现 BmDJ-1 在第 5 龄幼虫至成虫发育阶段具有独特的表达模式。我们使用鱼藤酮（ROT）在第 3 天 5 龄幼虫中评估 BmDJ-1 的抗氧化功能。在第 3 天 5 龄幼虫中给予 ROT 并提前 4 天用 BmNPV-BmDJ-1 感染外源 BmDJ-1，与对照组相比，BmDJ-1 组的 24 小时死亡率显著降低。2D-PAGE 显示，在 ROT 刺激下，BmN4 细胞中的 BmDJ-1 等电点（pI）向酸性形式转移。在所检查的影响血液 BmDJ-1 表达水平的因素中，根据明显的发育阶段依赖性变化，确定了一氧化氮（NO）浓度。与对照组相比，向 BmN4 细胞给予硝酸异山梨酯（ISDN），即 NO 供体，可增加 BmDJ-1 的表达。这些结果表明，BmDJ-1 可能由于 NO 而控制细胞中的氧化应激，并作为家蚕的发育调节因子。
