MuRF-GFP转基因斑马鱼模型的生成，用于研究Smyd1b和Hsp90α1敲低胚胎中的murf基因表达和蛋白定位。,Comparative Biochemistry and Physiology B: Biochemistry & Molecular Biology

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MuRF-GFP转基因斑马鱼模型的生成，用于研究Smyd1b和Hsp90α1敲低胚胎中的murf基因表达和蛋白定位。
Comparative Biochemistry and Physiology B: Biochemistry & Molecular Biology ( IF 1.9 ) Pub Date : 2019-10-24 , DOI: 10.1016/j.cbpb.2019.110368
Baojun Li ₁ , Siping Li ₂ , Qiuxia He ₂ , Shaojun Du ₂

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肌肉特异性RING手指蛋白（MuRF）是E3泛素连接酶，在骨骼肌和心肌蛋白质量控制中起重要作用。在这里，我们表征了斑马鱼胚胎中的murf基因表达和蛋白质定位。我们发现斑马鱼基因组包含六个murf基因，包括murf1a，murf1b，murf2a，murf2b，murf3和murf2样基因，它们在斑马鱼胚胎的骨骼肌和心肌中特异性表达。为了分析亚细胞定位，我们生成了表达MurF1a-GFP或MuRF2a-GFP融合蛋白的转基因斑马鱼模型。MuRF1a-GFP和MuRF2a-GFP显示出不同的亚细胞定位模式。MuRF1a-GFP在肌纤维中显示出横纹的定位模式，而MuRF2a-GFP主要表现出点状分布的随机图样。MuRF1a-GFP信号显示为沿着骨骼肌纤维中肉瘤M线对齐的小点。为了确定降低肌球蛋白蛋白降解的smyd1b或hsp90α1敲低是否可以改变murf基因表达或MuRF蛋白的定位，我们敲低了野生型smyd1b或hsp90α1的Tg（ef1a：MurF1a-GFP）和Tg（ef1a：MuRF2a-GFP）转基因斑马鱼的胚胎。击倒smyd1b或hsp90α1对murf基因表达没有影响。但是，MuRF1a-GFP的肌节分布在敲低的胚胎中被消除了。这伴随着斑马鱼胚胎肌肉细胞中MuRF1a-GFP随机点状分布的增加。总的来说，这些研究表明，MuRFs在斑马鱼胚胎发育的肌肉中特异性表达。

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Generation of MuRF-GFP transgenic zebrafish models for investigating murf gene expression and protein localization in Smyd1b and Hsp90α1 knockdown embryos.

Muscle-specific RING-finger proteins (MuRFs) are E3 ubiquitin ligases that play important roles in protein quality control in skeletal and cardiac muscles. Here we characterized murf gene expression and protein localization in zebrafish embryos. We found that the zebrafish genome contains six murf genes, including murf1a, murf1b, murf2a, murf2b, murf3 and a murf2-like gene that are specifically expressed in skeletal and cardiac muscles of zebrafish embryos. To analyze the subcellular localization, we generated transgenic zebrafish models expressing MurF1a-GFP or MuRF2a-GFP fusion proteins. MuRF1a-GFP and MuRF2a-GFP showed distinct patterns of subcellular localization. MuRF1a-GFP displayed a striated pattern of localization in myofibers, whereas MuRF2a-GFP mainly exhibited a random pattern of punctate distribution. The MuRF1a-GFP signal appeared as small dots aligned along the M-lines of the sarcomeres in skeletal myofibers. To determine whether knockdown of smyd1b or hsp90α1 that increased myosin protein degradation could alter murf gene expression or MuRF protein localization, we knocked down smyd1b or hsp90α1 in wild type, Tg(ef1a:MurF1a-GFP) and Tg(ef1a:MuRF2a-GFP) transgenic zebrafish embryos. Knockdown of smyd1b or hsp90α1 had no effect on murf gene expression. However, the sarcomeric distribution of MuRF1a-GFP was abolished in the knockdown embryos. This was accompanied by an increased random punctate distribution of MuRF1a-GFP in muscle cells of zebrafish embryos. Collectively, these studies demonstrate that MuRFs are specifically expressed in developing muscles of zebrafish embryos. The M-line localization MuRF1a is altered by sarcomere disruption in smyd1b or hsp90α1 knockdown embryos.

更新日期：2019-10-24

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