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標題: 白腹叢蚊原酚氧化酵素I基因之選殖與啟動子調控分析
Molecular Cloning and Promoter Regulation of Prophenoloxidase I Gene from the mosquito, Armigeres subalbatus
關鍵字: 原酚氧化酵素I;prophenoloxidase I;啟動子調控;promoter regulation
出版社: 昆蟲學系
昆蟲黑化包囊反應(melanotic encapsulation)是蚊子對抗外來入侵物的有效防禦機制,此反應之關鍵酵素為原酚氧化酵素 (prophenoloxidase; pro-PO)。本研究則利用基因庫篩選法,成功選殖到約2.4 kb的白腹叢蚊 (Armigeres subalbatus)原酚氧化酵素I基因(As-pro-PO I gene),並定序其核苷酸序列,發現此基因包含三個內插子 (intron),其長度大小分別是60、72及57 bp;在基因上游未轉錄區推定出多個與哺乳類動物急性免疫反應相同之調控因子,例如NF-κB (nuclear factor kappa B)、NF-IL 6 (nuclear factor interleukin 6)、ICRE (interferon consensus response element)、HNF-5 (hepatic nuclear factor 5)、NF-ELAM 1 (nuclear factor-endothelial leukocyte adhesion molecule 1)以及GATA factor。我們將As-pro-PO I上游約1,166 bp序列進行啟動子活性分析,得到NF-κB motif有可能是調控因子,接著利用電泳位移分析法(electrophorotic mobility shift assay, EMSA)和site-directed mutagenesis方法,證明人類p50 NF-κB細胞核蛋白與白腹叢蚊細胞核萃取的蛋白質皆可有效地結合在As-pro-PO I基因之推定NF-κB motif上,且其所結合的細胞核蛋白質的量會因蚊子進行黑化微絲蟲(microfilariae)而增加;此外,白腹叢蚊在注射NF-κB抑制劑後24小時,可顯著降低微絲蟲在白腹叢蚊體內的黑化度以及As-pro-PO I mRNA之表現。以上研究顯示NF-κB motif是白腹叢蚊原酚氧化酵素I基因重要的調控子。

Melanotic encapsulation is an effective defense reaction of mosquitoes against invading pathogens. The key enzyme in the biosynthesis of melanin in insects is prophenoloxidase (pro-PO). In this study, Armigeres subalbatus prophenoloxidase I (As-pro-PO I) gene, 2.4 kb, was cloned from DNA genomic library. As-pro-PO I gene contains three introns, i.e. 60, 72, and 57 bp in length from 5' end. Several nucleotide sequence regions homologous to mammalian regulatory motifs involved in the control of acute phase response genes, such as nuclear factor kappa B, nuclear factor interleukin 6, interferon consensus response element, hepatic nuclear factor 5, nuclear factor-endothelial leukocyte adhesion molecule 1, and GATA factors, were identified in the 5' untranscription region of As-pro-PO I gene. The results of cell transfection assay suggested that the NF-κB motif could be the regulatory element of As-pro-PO I gene. Subsequently, electrophoretic mobility shift assay (EMSA) and site-directed mutagenesis demonstrated that human NF-κB p50 protein and nuclear protein extract from mosquitoes can bind to NF-κB motif of As-pro-PO I gene. The binding activity of mosquito nuclear protein extract to NF-κB motif was increased after challenging with microfilariae. Also, an NF-κB inhibitor was inoculated into mosquitoes to elucidate the role of NF-κB-like protein in the regulation of As-pro-PO I gene. The degree of melanization of microfilariae in mosquitoes and the transcripts of As-pro-PO I were significantly decreased in NF-κB-inhibited mosquitoes. These data showed that NF-κB motif was an important regulation element of the As-pro-PO I gene.
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