豐年蝦蛋白激酶JAK與轉錄因子STAT基因表現與特性分析

Abstract

在本論文中，我們選殖出豐年蝦的蛋白激酶JAK與轉錄因子STAT，經由胺基酸的序列比對，豐年蝦與果蠅的蛋白激酶，雖然只有19%的相同性及33%的相似性，但是包含有完整的七個蛋白激酶保留性區域(JAK homology domain, JH domain)。另外，豐年蝦與果蠅的轉錄因子，則有較高的30%相同性。由這些低相同性的結果推測，無脊椎動物中不同的物種，在其蛋白激酶與轉錄因子具有其特異性。RT-PCR分析的結果顯示，豐年蝦蛋白激酶與轉錄因子在發育過程中呈現持續性的表現，與果蠅的蛋白激酶與轉錄因子在發育過程中表現特性相同。另外，我們也將豐年蝦蛋白激酶的JH1區域接合到轉錄因子的碳端(C-terminal)末端，構築了持續性活化的轉錄因子，AfSTAT-JH1, 可自行活化其酪胺酸處(tyrosine residue)，進而具有結合到果蠅Raf啟動子上的轉錄因子特定結合區域的能力。在SF9昆蟲細胞中，包含豐年蝦蛋白激酶和轉錄因子都具有轉錄活化(transactivation)果蠅Raf和totA啟動子的能力。然而，酪胺酸處磷酸化的活化態豐年蝦轉錄因子並未偵測到，經由細胞原位分析實驗顯示，大部份的豐年蝦轉錄因子都停留在細胞質中。我們的結果證實，包含蛋白激酶和轉錄因子都存在於豐年蝦的基因體中，將來對於研究JAK/STAT訊號傳遞路徑在豐年蝦的發育與免疫反應，提供了一個重要的基礎。

In this study, we isolated and characterized both JAK and STAT genes from Artemia, Artemia franciscana. Although AfJAK showed only 19% identity (33% similarity) to the Drosophila Hop protein, AfJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7. On the other hand, AfSTAT showed higher identity (30%) to Drosophila STAT (STAT92E). The low identities of AfJAK and SfSTAT to Drosophila Hop and STAT92E suggest that JAK and STAT proteins are unique in each different species of invertebrate. RT-PCR analysis showed that both AfJAK and AfSTAT transcripts were ubiquitously expressed in the embryo, which is similar to the expression patterns of Drosophila Hop and STAT92E mRNAs during development. In addition, we generated a constitutively active form of AfSTAT by fusing the JH1 domain of AfJAK to the C-terminal end of AfSTAT. This fusion protein, AfSTAT-JH1, autophosphorylated on its tyrosine residue and was able to bind to specific DNA motifs including the STAT-binding motifs in the Drosophila Raf promoter. Both AfJAK and AfSTAT proteins elicited the transactivation potential toward the fly Raf and totA promoter in Sf9 cells. However, tyrosine phosphorylation of AfSTAT was not detected, which is consistent with the cellular localization analysis that most AfSTAT proteins were in the cytoplasm. Our results demonstrate that both JAK and STAT are present in the genome of Artemia, which can serve as the basis for further investigations to explore the role of the JAK/STAT signal pathway in the development and immune response of brine shrimp.