以 GFAP (glial fibrillary acidic protein) intron 2 與 exon 1 序列當作研究靈長目與鯖亞目相近物種親緣關係的核標記

Abstract

近年來常以粒線體 DNA 和微衛星體序列作為研究物種演化與族群結構等課題之分子標記，但適合用於區分相近物種的分子標記卻還沒有被提出。本實驗室先前研究發現，利用細胞骨架之一的中間絲蛋白質（intermediate filaments, IFs）之基因序列建構無脊椎動物與脊椎動物之親緣關係圖時，無脊椎動物可與脊椎動物分開，且脊椎動物內部可依不同類型的 IF 各自分群，在此分群中又可看出不同物種間的演化關係，顯示 IF基因演化與物種演化具有相關性。本篇研究選擇第三型中間絲蛋白質 GFAP (glial fibrillary acidic protein)，因其專一表現在中樞神經系統，認為受於外界環境干擾也較小，其演化速度較能維持中性 (neutrality)。為了證實 GFAP 適合作為分子標記，我們從 Ensembl 資料庫中搜集 19 個物種的GFAP 序列，利用貝葉氏導出式 (Bayesian inference, BI) 分析親緣關係，結果顯示物種分群符合現今所接受分類，也確定了 GFAP 作為分子標記的可用性。由於 introns 具有大量核苷酸變化，以及可利用前後端 exons 保守序列設置引子，利於序列增幅等特性。近年來，許多利用 introns 進行物種分類的研究也日益增加。2010 年，Igea 等人將哺乳動物：人類、黑猩猩、獼猴、狗與牛基因體introns 序列，經由一連串篩選程序，找出隨機分散於基因體的 224 個 nuclear introns，成功利用此序列將五種相近物種區分。因此，本篇研究也期望能在GFAP 中，找出具有此特性的introns 片段。我們將人類、黑猩猩、大猩猩、長臂猿與獼猴五種靈長目物種的 8 個 GFAP introns 進行比對，發現 GFAP 的 intron 2 片段在五種靈長目物種中，長度皆等長。此外，利用該序列進行UPGMA 法親緣關係圖建構，亦能顯示出與其他研究結果一致的親緣遠近關係。於是我們選用了 intron 2 片段作為分子標記。由於傳統哺乳動物的分類是以化石證據為標準，相反地，魚類的分類卻是根據現生物種形態上的異同為主。為了統整兩個不同分類標準，我們以 GFAP 的 intron 2 片段，進行相近魚種的親緣關係研究。本篇研究選擇 4 科 9 屬 13 種的鯖亞目魚類，以PCR 技術將其 GFAP 的 intron 2 片段放大，定序完成後，進行序列長度與變異度比較。結果顯示不同科的魚種，其 intron 2 的長度皆不相同。但鯖科的鮪屬 (金槍魚屬) 和土魠魚 (馬鮫屬)，其 intron 2 長度卻與同是鯖科的花腹鯖 (鯖屬) 和圓花鰹（舵鰹屬）不一樣。隨後我們利用鯖亞目的 intron 2 序列，以 MP (最大簡約法) 與BI 譜系分析，結果顯示四 種鯖科魚類與帶魚科魚類為同一群組。此外，鯖科中的花腹鯖、圓花鰹彼此關係較近，並與另外同是鯖科的鮪屬、馬鮫屬分開。綜上所述，我們除了得知鯖科與帶魚科的親緣關係較近外，也建議鮪屬和馬鮫屬，應與花腹鯖、圓花鰹兩群組，重新分類為不同科物種較為適當。另外，我們加入 GFAP 基因中，可編錄蛋白質的片段 exon 1 進 行比對，結果顯示 exon 1 演化速率較 intron 2 快，可用於細究相近魚類物種間親緣關係的演變歷程。總結來說，本研究成功驗證了 GFAP intron 2 與 exon 1 片段作為分子標記的可用性，並以此重新檢視哺乳類與魚類的分類系統，也顯示GFAP 基因可作為區分相近物種的分子標記。

In recent years, mitochondrial DNA and microsatellite sequences are often used as the molecular markers for studying species evolution and population structure. However, to date, appropriate molecular markers for distinguishing closely related species have not been well-established. Our previous study demonstrated that the gene sequences of intermediate filaments (IFs) proteins, one of the major types of cytoskeleton, could separate vertebrates and invertebrates into two different groups in phylogenetic reconstruction. In addition, each of the vertebrate IF genes was separated into different subgroups corresponding to their IF types. Furthermore, within each subgroup, the evolutionary relationship of different species is parallel to that of species. These results suggested that the evolution of IF genes is closely related to the evolution of vertebrate species. In this study, the gene of type III intermediate filament protein GFAP (glial fibrillary acidic protein) was selected as the molecular marker. Due to its specific expression in the central nervous system, it is postulated that its selective pressure from the external environment is minimum, thus it is more likely to maintain neutral. To prove that the GFAP gene is an appropriate molecular marker, we collected the GFAP sequences of each species from the Ensembl database. Subsequently, we generated the phylogenetic tree of the GFAP gene sequences of 19 animal species by Bayesian inference (BI). The results was in agree with the currently accepted classification. Therefore, the validity of GFAP gene as a molecular marker is confirmed. Recently, it has been shown that introns have relatively large nucleotide variability, and can be easily amplified with primers placed in the adjacent exons. Thus, there are increasing number of studies using intronic sequence to investigate the phylogenetic relationship of species. In 2010, Igea et al., by applying several reasonable filters, selected 224 intronic sequences that belongs to several mammalian species, including human, chimpanzee, macaque, dog and cattle, and successfully distinguish the phylogenetic relationship of these species. Therefore, in this study, it is anticipated to characterize the appropriate introns of GFAP gene for distinguishing the closely related species. By aligning the 8 intronic sequence of GFAP gene of five primate species, including human, chimpanzee, gorilla, gibbon and macaque, it was revealed that the length of the intron 2 fragment are the same among five primate species. In addition, the phylogenetic tree of these five primate species reconstructed with GFAP intron 2 by UPGMA method was also in consensus with currently established phylogenetic relationship. Thus, the intron 2 sequence was selected as the molecular marker for the current study. The traditional classification for mammals is based on fossil evidence. On the other hand, classification of fishes is mainly based on morphology of the extant species. Thus, the aim of this study is to examine the phylogenetic relationship of species under a unified standard by examining GFAP intron 2 sequences of primates and fishes. In our study, fishes of several different families and genera in the suborder Scombroidei were selected, and their GFAP genes sequences were amplified by PCR. After sequencing, their lengths and variation were compared. The results revealed the length of GFAP intron 2 is different in each of the five fish families. Surprisingly, although from the same family of Scombridae, the length of GFAP intron 2 of the yellowfin tuna, bigeye tuna, albacore tuna,, Pacific bluefin tuna (Thunnus) and the seer fish (Scomberomorus) is distinct from that of spotted mackerel (Scomber) and frigate mackerel (Auxis). Then, we reconstructed the phylogenetic tree by intron 2 sequence from each of the fishes, the results revealed one single group among the four fishes of Scombridae and fishes of Trichiuridae. Moreover, among the Scombridae, spotted mackerel and frigate mackerel are closer to each other, but are distinct from the Thunns and seer fish. Taken together, the results suggest that the evolutionary relationship of Scombridae and Trichiuridae are closer than the currently accepted phylogenetic classification. Furthermore, it is also proposed that spotted mackerel, frigate mackerel should be separated from Thunns, seer fish, and regrouped into a new family. Besides, the protein coding region of GFAP exon 1 was also included in our analysis. The results showed that the evolutionary rate of exon 1 is higher than intron 2, thus it can be used to clarify the phylogenetic relationship of closely related fish species. In summary, the current study confirmed the validity of GFAP intron 2 and exon 1 as the molecular marker for closely related species. We have also re-examined the phylogenetic relationship of mammals and fishes, and the results shows that GFAP may have the potential to serve as one of the species barcode genes.