- 學位論文
斑馬魚抑制性肌鈣蛋白分子結構、發育動態表現及啟動子之分析
The Molecular Structures, Expression Patterns, and Promoter Analysis of Zebrafish Troponin I Genes
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摘要
抑制型肌鈣蛋白(TnnI)是位於橫紋肌細肌絲中對細肌絲的收縮提供鈣離子轉換的一個肌鈣蛋白複合體組成。臨床上在急性冠狀動脈病徵下，心臟型TnnI在脊椎動物中被當作具有高敏感性及專一性的指標。 雖然在鳥類及哺乳類中，已經確立存在三種不同異購蛋白的TnnI，分別是快肌型、心肌型、及慢肌型。但在低等脊椎動物中，對於TnnI基因的了解並未十分透徹。為了更了解在低等脊椎動物中TnnI在發育調控上所扮演的地位。首先，我們先區別了斑馬魚的TnnI基因，TnnI1及TnnI2。在脊椎動物TnnI的基因演化樹中，TnnI1及TnnI2分別相近於脊椎動物的TnnI1(slow muscle)及TnnI2(fast muscle)。在反轉錄聚合酶連鎖反應及全胚胎原位雜交法來進行觀察，結果顯示出在受精後16小時，TnnI1及TnnI2會開始表現在斑馬魚體節的部位，此外在軀幹橫切顯示出TnnI1及TnnI2是分別專一表現在慢肌及快肌，藉由這些結果來分別定義了TnnI1及TnnI2分別為斑馬魚慢肌型TnnI及快肌型TnnI。另外我們也定義了一個新的TnnI 異構型蛋白，TnnI-HC，我們觀察到TnnI-HC會表現在早期的cardiogenesis (16 hpf)，且會持續在心臟專一性表現。有趣的是，在48hpf之後便會開始在頭部肌肉及心臟表現，雖然TnnI-HC較相似於慢肌型 TnnI，但卻會在心臟及頭部肌肉中有表現，這種TnnI異構型蛋白表現情形是不同於其他高等脊椎動物。所以我們利用斑馬魚基因庫來篩選TnnI-HC大約7.6-Kb的上游片段，藉由此片段可以來分析TnnI-HC在斑馬魚中的發育調控。我們選殖了兩段斑馬魚TnnI-HC上游序列1.4-Kb (-1400/+30)及intron 1 4-Kb (+2257/+6253)，並分別構築了EGFP表現質體並進行顯微注射。我們發現到1.4-Kb的上游序列並沒有啟動子的能力，然而當注射intron 1 4-Kb，能觀察到intron 1 4-Kb能夠獨自驅動EGFP在心臟及軀幹中表現。由此我們假設在TnnI-HC其他上游序列中仍有其他位置能夠來加強頭部肌肉的表現及抑制體節的表現。利用deletion analysis來分析intron 1 4-Kb的序列後。我們可以定義在intron 1 (+2257/+4689)中，具有一個cis-element能夠來調控早期斑馬魚TnnI-HC基因在心臟中的表現。
並列摘要
Troponin I (TnnI), a constituent of the troponin complex on the thin filament, provides a calcium-sensitive switch for striated muscle contraction. Cardiac TnnI is a highly sensitive and specific marker of myocardial injury in the acute coronary syndromes. Although TnnI gene has been identified in birds and mammals as it encodes the isoforms expressed in cardiac muscle, fast skeletal muscle and slow skeletal muscle, there is extremely little known about the developmental regulation of TnnI gene in the lower vertebrates. Firstly, we characterized zebrafish TnnI genes, TnnI1 and TnnI2. In the unrooted radial gene tree for TnnI genes among vertebrates, the zebrafish TnnI1 and TnnI2 genes we cloned correspond to the slow muscle TnnI1 gene and the fast muscle TnnI2 gene of other vertebrates, respectively. Secondly, reverse transcription-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization showed that TnnI1 and TnnI2 transcripts were expressed in the somites at 16 hours post-fertilization (hpf). In addition, we also demonstrated that the expression of TnnI1 and TnnI2 genes was slow- and fast-muscle-specific, respectively, indicating that zebrafish TnnI1 and TnnI2 proteins were slow-TnnI and fast -TnnI, respectively. Interestingly, we also identified a novel TnnI isoform, TnnI-HC (heart and craniofacial), which was expressed in the heart exclusively during early cardiogenesis at 16 hpf, but it was not only in the craniofacial muscle but also in the heart after 48 hpf. Thirdly, in order to go further understanding the regulatory mechanism of TnnI-HC gene, we cloned a 7.6-kb fragment containing a partial TnnI-HC genomic gene from the zebrafish genomic library. Thereafter, we subcloned an upstream 1.4-Kb (from -1400 to +30; -1400/+30) and an intron 1 4-Kb (from +2257 to +6253; +2257/+6253) and we fused them with GFP separately. After microinjection, we found that the -1.4 Kb fragment did not contain any promoter activity. However microinjection of an intron 1 fragment (4-Kb) alone without including -1.4 Kb enabled the expression of GFP in the heart and trunk muscle, but not in the craniofacial muscle. Thus, we speculate that other upstream regulatory segment might play a role in the enhancing the craniofacial muscle expression and repressing the trunk expression of TnnI-HC gene. Deletion analysis in +2257/+6253 segment, we also identified a cis-element located at +2257/+4689 within intron 1 that might control the cardiac expression of zebrafish TnnI-HC gene during early development.
參考文獻
Baldwin, A. S. Jr., Kittler, E. L. W., & Emerson, C. P. Jr., (1985). Structure, evolution, and regulation of a fast skeletal muscle troponin I gene. Proceedings of the National Academy of Sciences.U.S.A. 82, 8080–8084.
Banerjee-Basu, S., & Buonanno, A., (1993). cis-Acting Sequences of the Rat Troponin I Slow Gene Confer Tissue- and Development-Specific Transcription in Cultured Muscle Cells as well as Fiber Type Specificity in Transgenic Mice. Molecular and cellular biology. 13, 7019-7028.
-mal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected TnI isoforms. Molecular and cellular biology. 13, 1433–1439.
Fabiaot, A., (1983). Calcium-induced release of calcium fron the cardiac sarcoplasmic reticulum. American journal of physiology 35, 245-250.
Farah, C. S., & Reinach, F. C., (1995). The troponin complex and regulation of