頭索動物文昌魚bHLH-PAS基因之研究

Abstract

bHLH-PAS轉錄因子廣泛存在於原口動物與後口動物各動物門，它們參與許多重要的發育與生理功能，包含中樞神經系統的區域分化、氣管生成、低氧感應、芳香烴感應，以及調控生物時鐘等。雖然它們的功能在無脊椎模式物種與脊椎動物之間有明顯的差異，目前對於這些基因功能的演化所知仍甚少。為瞭解這些基因在脊索動物的演化，我以頭索動物文昌魚 (Branchiostoma floridae) 作為材料，由既有的基因組和基因模型 (gene models) 中鑑別文昌魚的bHLH-PAS基因。我的結果顯示：文昌魚擁有至少十個bHLH-PAS基因，其中九個可以被歸類到已知的直系同源基因家族。再者，由獲得的cDNA與gene models之間的差異，可瞭解到既有的gene models無法完全正確地預測真實的基因序列。最後，我使用即時聚合酶鏈鎖反應與核酸原位雜交方法，檢驗了文昌魚胚胎發育過程中這些基因的表現模式，發現BfArnt、BfNcoa、BfSim、BfHifα等基因的表現模式與脊椎動物的同源基因表現模式非常相似，這可能表示它們的功能保守；但是另一些基因，如BfAhr與BfNpas4，其表現模式與脊椎動物同源基因有明顯差異，這可能表示這些基因的功能在兩個演化分支分歧之後發生改變。脊椎動物擁有多個旁系同源的bHLH-PAS基因，這些基因可能是來自脊椎動物的全基因組複製，我比較這些旁系同源基因的表現模式與功能，推論功能的分化或新功能的獲得可能可以解釋脊椎動物所擁有的多個旁系同源基因的功能歧異性。

The bHLH-PAS transcription factors are found in both protostomes and deuterostomes. They are involved in many developmental and physiological processes including the regional differentiation of the central nervous system, tube-formation, hypoxia signaling, aromatic hydrocarbon sensing, and circadian rhythm regulation. Although some of their functions are apparently different between model protostomes and vertebrates, little has been known about the evolution of their functions. To understand the evolution of these genes in chordates, I manually annotated bHLH-PAS genes of the basal chordate amphioxus (Branchiostoma floridae) from the draft genome database. My results show that amphioxus has at least 10 bHLH-PAS genes, 9 of which can be assigned to known orthologous families. Moreover, the comparisons of cDNA sequences and gene models show that current amphioxus gene models may not correctly predict the true coding sequence. Finally, I examined the developmental expression patterns of these amphioxus bHLH-PAS genes by quantitative polymerase chain reaction and in situ hybridization. I found that BfArnt, BfNcoa, BfSim, and BfHifα are expressed in similar patterns compared to their vertebrate homologues, suggesting that their functions may be conserved. On the other hand, BfAhr and BfNpas4 are expressed with different patterns between amphioxus and vertebrates. This might imply functional changes after the divergence of cephalochordates and vertebrates. Since vertebrates have multiple bHLH-PAS paralogues that may result from whole genome duplications, I compared the expression patterns and the functions of these paralogues. I suggest that neo- or subfunctionalization of the duplicated paralogues may explain the functional divergence of bHLH-PAS genes in the vertebrate lineage.