請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/30742
標題: The functional analysis of a guanylyl cyclase, BdmGC-1, in the oriental fruit fly, Bactrocera dorsalis (Hendel)
東方果實蠅(Bactrocera dorsalis (Hendel))鳥苷酸環化酵素BdmGC-1之功能分析
作者: 張哲誠
Chang, Jer-Cherng
關鍵字: 鳥苷酸環化酵素
出版社: 昆蟲學系所
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摘要: 本研究主旨為分析一鳥苷酸環化酵素基因BdmGC-1於東方果實蠅(Bactrocera dorsalis (Hendel))(果實蠅科:雙翅目)之生理作用機制。試驗結果指出,此基因至少具兩個不同之異位截切之轉譯子(alternative splicing variants),分別命名為BdmGC-1與BdmGC-1B。而此二轉譯子所對應之蛋白質序列,顯示均具膜蛋白型鳥苷酸環酵素之特性,即由蛋白質N端往C端分別為訊息蛋白(signal peptide)、膜外受體區(extra-cellular ligand binding domain)、穿膜區域(transmembrane region)、類蛋白激酶調控區(regulatory kinase-homology domain)與鳥苷酸環化酵素活化位(cyclase catalytic region)。但此二蛋白有些許差異處,如BdmGC-1B於膜外受體區多了46個胺基酸之插入序列;而BdmGC-1則多出C端之尾部。當BdmGC-1於293T細胞表現,如預測顯示,其為膜外表現之糖基化蛋白,並明顯具鳥苷酸環化酵素特性;但BdmGC-1對已知鳥苷酸環化酵素配體(ligand)並無明顯反應。在基因表現上,BdmGC-1主要於蟲體發育期表達,而成蟲並無明顯表現。後續研究以蟲體進行專一性免疫標定與核酸原位雜合,BdmGC-1被發現於蟲體氣管上之氣門內腺(epitracheal gland)表達。而以羽化激素(eclosion hormone)分別對表現BdmGC-1或BdmGC-1B之細胞進行測試,得知表現BdmGC-1之細胞群對低濃度之蛻皮激素有強烈反應;而表現BdmGC-1B之細胞群僅對高濃度之蛻皮激素有反應。此結果顯示此二蛋白於東方果實蠅生理上所扮演之角色有所不同。
Two novel guanylyl cyclases BdmGC-1 and its spliced variant BdmGC-1B were isolated from the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera), and proposed to possess all the characteristics of receptor GC, i.e. a signal peptide, an extracellular ligand binding domain (ECD), a transmembrane region, a regulatory kinase-homology domain, and a core cyclase catalytic region. Notably, the spliced variant BdmGC-1B possesses an additional 46 amino acid insertion in the extracellular domain but lacks the C-terminal tail of BdmGC-1. In heterogeneous expression, BdmGC-1 is manifested as a cell-surface glycoprotein with marked cGMP-generating activity, but unresponsive to ligands for mammalian receptor GCs. The mRNAs of BdmGC-1 were found highly expressed in all of the developmental stages, i.e., embryo, larva and pupa, but found quite low or no expression in adult tissues. Immunolabeling and in situ hybridization revealed that BdmGC-1s are expressed in Inka cells of epitracheal glands. Heterologous expression of BdmGC-1 in HEK-293T cells leads to robust increases in cGMP following exposure to low picomolar concentrations of eclosion hormone (EH). The B-isoform responds to higher EH concentrations, suggesting different physiological roles of these two cyclases. BdmGC-1 and BdmGC-1B are proposed as high- and low-affinity EH receptors, respectively.
URI: http://hdl.handle.net/11455/30742
其他識別: U0005-1908200910424500


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