斑馬魚早期胚胎之蛋白質體之研究

Abstract

在母體生成卵子的過程中，母體會製造核醣核酸和蛋白質儲存在成熟的卵子內，這些核醣核酸和蛋白質就稱為母體因子。在脊椎動物的胚胎發育過程中，受精後的胚胎基因組(zygotic genome)並未能在一開始就立即的被活化且進行作用，因此母體因子主導胚胎最初期的發育過程。在不同的物種中，母體因子對於胚胎發育的影響也有所不同。較為低等的動物，譬如無脊椎動物(果蠅、線蟲)，或是低等脊椎動物(蛙)等，對於母體因子的依賴時間較長，直到胚胎分裂至數千個以上的細胞時，受精後胚胎基因組才會被活化。以斑馬魚的胚胎發育過程來說，其胚胎基因組約在胚胎分裂至一千個細胞(約3小時)左右時開始表現。在此時期之後，母體因子對於胚胎的控制會逐漸地被胚胎基因體所表現的因子所取代，因此，此時期又稱為母體轉換合子的過渡時期 (MZT)。在受精之後，這些母體因子啟動胚胎發育的程式來完成胚胎的生長與分化。為了能夠了解這些母體因子在胚胎發育時期的變化，我們利用蛋白質體學的方式來分析斑馬魚早期胚胎發育，特別針對受精前後(卵母細胞轉換胚胎；(OET))以及母體轉換合子(MZT)的兩個過渡時期之中，蛋白質種類的變化。結合一維膠電泳(1D SDS-PAGE)與串聯式質譜法(LC-MS/MS)方法，我們鑑定出1109個蛋白質並依其生物功能分類。我們發現，在上述之胚胎時期，均能偵測到許多負責蛋白質轉譯與轉譯後修飾功能的蛋白質。結果顯示，母體核醣核酸在受精後，便立即被轉譯成蛋白質，以供後續發育過程使用。我們也發現在這兩個過渡時期中，一些調控細胞週期、胚胎發育以及胚胎基因體活化機制的蛋白質，其表現量會有增減的現象。將我們蛋白質體學的結果和之前在基因體學上研究的資料做比較，我們發現一些來自母體的蛋白質，其核醣核酸並沒有在這些時期被偵測到。也就是說，這些蛋白質和與其對應的核醣核酸在這些時期的表現量並不一致。這個現象更證實了蛋白質體學和基因體學資料互補的重要性。總而言之，我們提供了新的研究資料以便篩選出在脊椎動物早期胚胎發育的過程中，具有調控性的重要蛋白質，藉此進行下一步的標的定量及更加精確的系統性遺傳學分析。

All processes that occur before the activation of the zygotic genome at midblustula transition are driven by maternally inherited products, which have been synthesized and stored in mature oocytes during oogenesis. After fertilization, these maternal factors initiate developmental cascades that carry out the embryonic developmental program. To investigate the dynamic expressed pattern of these maternal factors, total proteomic analysis was performed during two transitions, oocyte-to-embryo transition (OET) and maternal-to-zygotic transition (MZT), in zebrafish early development. Using 1D SDS-PAGE and LC-MS/MS, we identified 1109 proteins and categorized them based on their biological functions. Many proteins involved in translation or post-translational modification were actively expressed during the selected five stages. This result implied that maternal mRNAs required for subsequent developmental processes were translated rapidly under well control after fertilization. Some of the proteins with differential expression during OET or MZT are proteins involved in cell-cycle regulation, development processes, and zygotic genome activation. The dynamic expressions of these proteins assisted embryos in progressing these two important transitions and subsequent developmental program. A comparison of our proteomic results with transcriptomic data obtained in other studies revealed that some of the proteins are maternally deposited since no transcripts can be detected in these selected stages. In other words, these protein levels did not correspond to their mRNAs amounts during these stages. This result confirmed idea that proteomics or transcriptomics approach alone cannot reveal the complete picture about any biological process; instead, both approaches are complementary and essential for the description of biological events at molecular levels. In this study, we provide a new resource for the selection of candidate proteins for targeted quantitation and refined systematic genetic network analysis in vertebrate development in biology.