斑馬魚 Myostatin 基因之分子調控與功能分析

Myostatin gene regulation and functional assay in zebrafish

李秋月(Chiou-Yueh Lee)

指導教授 ： 吳金洌

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.00966

摘要

哺乳類動物肌肉倍增基因(Myostatin) 缺乏會造成骨骼肌增加及變大導致成 ”肌肉倍增” 表現型，Myostatin 在牛及人類主要表現在骨骼肌中，其突變或缺乏會造成骨骼肌細胞質量及數目的增加，是高等脊椎動物骨骼肌發育與生長之主要負調控因子；對魚類而言，myostatin可在多種組織中表現，如腦、鰓、心臟、卵巢、眼睛、腎臟等器官，故其並非只單純調控肌肉組織生長，可能同時參與多種組織之調控機制。本實驗則分別從斑馬魚基因體基因庫及 24 小時cDNA 基因庫中篩選基因體 DNA 及 cDNA ，分別探討 myostatin 基因之調控與功能。經選殖後得到7 個有訊號的基因體 DNA ，其中有三個選殖株為 myostatin I 基因組，分別為Zg 3-1的13.5Kb 、Zg 15-1的 24Kb 及 Zg 17-3的15Kb，其中以 15-1 包含有 myostatin I基因的啟動子及基因組在內，為了分析啟動子的表現，利用 PCR 增幅方式取得啟動子片段5.9Kb、5.18Kb、3.4Kb、2.85Kb、2.55Kb、1.69Kb 及 0.6Kb 等7 個片段，並將其構築至 pGL3-basic 冷光表現載體及pEGFP-1綠色螢光蛋白載體上，並將 pGL3-basic 冷光表現選殖株轉殖到 C2C12 、ZFL、NIH3T3 細胞中看其表現， 在 C2C12 及 ZFL 細胞中以 3.4Kb 長的啟動子有最大活性， NIH3T3 細胞表現情形則以 0.6 Kb 有最佳活性，將含 pEGFP-MSYN 綠色螢光蛋白選殖株利用顯微注射打入斑馬魚胚胎中，觀察 myostatin 啟動子在生物體之表現情形，結果並無預期之綠色螢光之表現。
另為瞭解myostatin之功能，由斑馬魚 24 小時胚胎所合成的 cDNA 庫所選殖出 myostatin II 並將 exon III 刪除 281 bp利用vector base-RNA方式將其構築至pEGFP-C1 載體，利用顯微注射打入斑馬魚胚胎中，取得一個遺傳穩定myostatin 基因knockdown 的肌肉倍增斑馬魚。結果在胚胎早期發育時為全身發亮，至72 小時開始往肝臟集中至 96 小時則表現於肝臟及脊椎且穩定綠色螢光表現於成魚中，經進一步交配後其子代仍可穩定且持續的表現。利用定量核酸聚合酶連鎖反應分析確認 RNA降低為野生組的30%，肌肉蛋白質含量降為野生組的36.76%；肌肉生成機制中相關標示因子 MyoD、Myogenin、 Mrf4 和 Myf5 之RNA增加為原來的2倍以上；分析第六代的成長速率，發現在4個月大時， 其野生組和轉基因組兩組間平均體重0.38 ± 0.05 公克及0.55 ± 0.11 公克， 轉基因組體重增加了45%；經 t-test 統計分析兩組間彼此有顯著差異， 進一步將一個月的稚魚切片及經 HE 染色分析肌肉纖維細胞大小由野生組平均值230.69 ± 139.04 μm2 增加至轉基因組之428.97 ± 207.2 μm2 平均增加了 85.95％，且兩者間具有顯著差異(p<0.05)。在本實驗中針對斑馬魚 myostatin 進行基因沉默 (gene silence) 可得到穩定遺傳世代表現之轉殖基因魚，這是首次成功得到具有 "double muscle" 表現型特徵的模式斑馬魚。

關鍵字

斑馬魚 ； 肌肉倍增基因 ； 啟動子 ； 基因靜默 ； 基因轉殖魚

並列摘要

Myostatin belongs to the transforming growth factor (TGF)-β superfamily, and is a secreted growth and differentiation factor and also a potent negative regulator of skeletal muscle development and growth. The myostatin (MSTN) null phenotype in mammals were characterized by extreme gains in skeletal muscles mass or “double muscle” as the cytokine negatively regulates skeletal muscle growth and development. Several aspects of MSTN biology in the fish were quit different from that in mammals. MSTN homologs had been identified in many different fish species. This diverse patter of expression in fish suggests that the biological actions of MSTN may not be restricted to skeletal muscle but may additionally influence other fish tissues as well. In this study, we had establish the zebrafish genomic library and obtained seven lambda clones by using the zebrafish MSTN I cDNA as probe. After subcloning, analysing and comparison sequence with the documented MSTN I cDNA, we found that Zg3-1 clone included 12.5Kb promoter region and exon I, Zg15-1 including 11.5 Kb promoter, exon I, intron I, exon II, intron II, exon III, intron III and 3’ untranslation region. To analyze the promoter activity of myostatin I gene, we ligated 7 different DNA fragments of the myostatin I gene into pGL3-basic vector and constructed plasmids pMSTN5.9K, pMSTN5.18K, pMSTN3.4K, pMSTN2.85K, pMSTN2.55K, pMSTN1.69K and pMSTN0.6K. As transfection to C2C12, ZFL (zebrafish liver cell) and NIH3T3 cells, the expression of luciferase activity was analysed. The results indicate that there were 12 E-boxes present in the 3.4Kb fragment of the zebrafish myostatin promoter, E11 and E12 E-box plays an important role in the regulation of the promoter activity in C2C12 and ZFL cells.
We utilized microinjection of an antisense RNA-expressing vector to establish a myostatin gene knockdown hereditarily stable zebrafish strain with a double-muscle phenotype. Through real time-PCR and immunostaining analysis, the myostatin messenger (m)RNA and protein levels of homozygous transgenic zebrafish were only 33% and 26% those of the non-transgenic control, respectively. And the mRNA level of myogenic regulatory factor markers, such as MyoD, myogenin, Mrf4, and Myf5 were dramatically increased in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish showed 45% heavier body weights than those of non-transgenic controls. Histochemical analysis of 1-month-old fry showed that the area of the cross-section of homozygous transgenic fish muscle fiber was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.

並列關鍵字

zebrafish ； myostatin ； gene silence ； promoter ； transgene fish

參考文獻

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