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廖永冠,李育媚,鲍宝龙.2021.opn1lw2基因在红光诱导斑马鱼皮肤 色素细胞形成中的作用.动物学杂志,56(4):608-616.
opn1lw2基因在红光诱导斑马鱼皮肤 色素细胞形成中的作用
The Role of opn1lw2 in the Formation of Skin Pigment Cells in Zebrafish (Danio rerio) under Red Irradiation
投稿时间:2021-01-07  修订日期:2021-05-18
DOI:10.13859/j.cjz.202104013
中文关键词:  色素细胞  红光  opn1lw2  斑马鱼  视黄酸合成酶
英文关键词:Pigment cells  Red light  opn1lw2  Zebrafish  Retinoic acid synthase
基金项目:国家自然科学基金项目(No. 31872546)
作者单位E-mail
廖永冠 上海海洋大学 949790185@qq.com 
李育媚 上海海洋大学 1092014473@qq.com 
鲍宝龙 上海海洋大学 blbao@shou.edu.cn 
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中文摘要:
      为了探究感红光视蛋白2基因opn1lw2在红光诱导斑马鱼(Danio rerio)皮肤色素细胞形成中的作用,针对AB品系野生型斑马鱼利用CRISPR/Cas9基因编辑技术敲除感红光视蛋白2基因opn1lw2,构建opn1lw2缺失的纯合opn1lw2-/-品系。使用光强(800 ± 100)lx的红光LED灯(每天光照24 h)对15日龄野生型斑马鱼和opn1lw2-/-品系斑马鱼进行60 d水面照射,发现野生型斑马鱼背部皮肤黑色素细胞数量显著多于opn1lw2-/-品系斑马鱼。实时荧光定量PCR分析发现,黑色素细胞标记基因kit在野生型斑马鱼背部皮肤表达量显著高于opn1lw2-/-品系,黄色素细胞标记基因csf1ra和虹彩细胞标记基因pnp4a在opn1lw2-/-品系及野生斑马鱼背部皮肤表达无显著差异。表明红光能通过opn1lw2基因调控斑马鱼背部皮肤黑色素细胞的形成,但不影响皮肤黄色素细胞和虹彩细胞的形成;而且,调控黑色素细胞分化的α-MSH促黑激素的前体基因pomca在红光持续照射60 d的opn1lw2-/-品系斑马鱼背部皮肤中的表达显著低于野生型,表明红光通过opn1lw2基因调控pomca基因的表达从而诱导黑色素细胞的形成。实时荧光定量PCR检测发现,野生型斑马鱼皮肤中视黄醛脱氢酶基因raldh3表达量显著高于opn1lw2-/-品系,而视黄醛脱氢酶基因raldh2的表达,在两种类型斑马鱼中没有差异,表明opn1lw2基因可介导红光诱导视黄醛脱氢酶基因raldh3表达,进而调控黑色素细胞的形成。这些结果对于深入理解红光诱导鱼类皮肤色素细胞形成有重要帮助。
英文摘要:
      In order to explore the effect of red light on the formation of skin pigment cells, CRISPR/Cas9 gene editing technology was used to construct a zebrafish line with homozygous deletion of the red light opsin 2 gene opn1lw2. A red LED lamp with a light intensity of 800 ± 100 lx was used to irradiate 15-day-old AB strain wild-type zebrafish (Danio rerio) and opn1lw2-/- strain zebrafish for 60 days, the number of melanocytes in the dorsum skin of wild-type and mutant zebrafish were counted with Photoshop CS5,and the data were analysed using GrahPad Prsim6. The results showed that the number of melanocytes in the dorsum skin of wild-type zebrafish was significantly more than that of the mutant (Fig. 2a﹣c). Real-Time quantitative PCR (RT-PCR) analysis found that the expression of the melanocyte marker gene kit in the dorsum skin of wild-type zebrafish was significantly higher than that of the mutant (Fig. 2d). There was no significant difference in the expression of the xanthophore marker gene csf1ra and the iridophores marker gene pnp4a of the dorsum skin between wild zebrafish and the opn1lw2-/- mutant (Fig. 3c, d), indicating that red light can regulate the formation of melanocytes in the dorsum skin of zebrafish through the opn1lw2, but does not affect the formation of skin xanthophores and iridophores; moreover, it regulates the differentiation of melanocytes. The expression of the precursor gene pomca of α-MSH in the dorsum skin of opn1lw2-/- zebrafish was significantly lower than that of the wild type (Fig. 2e), indicating that red light regulates the expression of pomca gene through opn1lw2 to induce the formation of melanocyte; RT-PCR detection found that the expression of retinal dehydrogenase raldh3 in wild-type zebrafish skin was significantly higher than that of mutant opn1lw2-/- (Fig. 4b), while there was no difference in expression of raldh2 encoding retinal dehydrogenase (Fig. 4a), indicating that opn1lw2 may selectively mediate the red light-induced synthesis of retinoic acid to regulate the formation of melanocytes. These results are importantfor understanding the formation of pigment cells in fish skin induced by red light irradiation.
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