梨形鞭毛蟲的WRKY蛋白質對於不同基因的轉錄調控之影響

Abstract

WRKY蛋白質在植物中是一個大型的DNA結合蛋白質家族，可調控許多細胞的生理功能，與生長、發育、疾病的抵抗、壓迫反應及衰老等作用有關。 我們從梨形鞭毛蟲的基因體中找到一個wrky基因，經由序列分析發現是具有兩個WRKY domain，屬於Group I WRKY。我們也發現wrky基因的mRNA量在囊體化時期比在滋養體時期增加。我們將HA標記接到WRKY並轉染梨形鞭毛蟲，利用免疫螢光染色發現WRKY蛋白質在滋養體時期是位於整個細胞中，包含細胞核與細胞質，在囊體化則只有出現在細胞核中。 EMSA實驗中也發現WRKY蛋白質可和wrky本身的啟動子結合，也可以和ER stress marker bip基因的啟動子結合。經由突變序列分析，也發現WRKY蛋白質的結合序列為g/aGTCAg/a，與植物WRKY蛋白質的結合序列相似。我針對梨形鞭毛蟲的38個基因啟動子區域進行搜尋，發現有13個基因含WRKY的結合位置，其中包含cyst wall protein 2 (cwp2)基因，經由EMSA實驗結果也發現WRKY可和cwp2基因的啟動子結合。另外，當大量表現WRKY時會使cwp2基因的mRNA量增加，推測WRKY對cwp2基因的調控可能是作為轉錄活化子。 我們也利用螢光酵素基因接上wrky或是bip基因啟動子，如果將此兩基因啟動子上的WRKY結合位置突變，也發現啟動子活性有明顯的下降，顯示這些WRKY蛋白質的結合位置是positive cis-acting elements，推測WRKY對wrky基因本身可能有positive autoregulation，對bip基因的調控可能是作為轉錄活化子。另外，我們發現在加入ER stress inducer DTT後，wrky基因的mRNA量及啟動子活性皆有明顯的上升，和之前實驗室同學發現加入DTT後，bip基因的mRNA量有明顯的上升相似，我也發現bip基因啟動子活性在加入DTT有明顯的上升。有趣的是，DTT也會造成WRKY蛋白質進入細胞核中。另外，當大量表現WRKY時，會使bip基因的mRNA量增加，推測WRKY對bip基因的調控可能是作為轉錄活化子。 我們也發現WRKY蛋白質可和cyclin b基因啟動子的aTTCAa序列結合，此序列與已知的WRKY結合序列g/aGTCAg/a相似，當大量表現WRKY時會促進cyclin b基因的mRNA量增加，推測WRKY蛋白質對cyclin b基因的調控可能是作為轉錄活化因子。 由實驗結果可得知，WRKY蛋白質對於梨形鞭毛蟲而言，可能是重要的轉錄調節因子，且參與許多梨形鞭毛蟲基因的轉錄調控，包括ER stress相關基因，cyst wall protein基因，及cell cycle相關基因。

WRKY homologues constitute a large family of DNA-binding proteins in plants that may be needed for a variety of key cellular functions including disease-resistance, stress response, and development. A putative wrky gene has been identified in Giardia lamblia genome. After analysis of the amino acid sequence of WRKY protein, we found that it has two WRKY domains and belongs to group I WRKY protein. We also found that wrky mRNA levels increased slightly during encystation. The HA-tagged WRKY localized in the two nuclei and cytosol at vegetative stage, but it only localized to the two nuclei during encystation . Recombinant WRKY specifically bound to its own promoter and the ER stress response marker bip gene promoter. Mutation analysis revealed that an g/aGTCAg/a was required for binding of WRKY to these promoters. The antisense sequence of the WRKY binding site is similar to the sequence of plant WRKY binding sites, c/tTGACc/t, on the coding strand. We searched the 200-bp 5’-flanking regions of 38 Giardia gene, and found that 13 genes contain the WRKY binding site, including cyst wall protein 2 gene. We found that WRKY protein can bind to the cwp2 promoter, and that overexpression of WRKY protein resulted in an increase of the cwp2 mRNA level. Our results suggest that WRKY may be an important transcriptional activator for cwp2 gene. Using mutated plasmids and transfection assays, we demonstrated that one of the WRKY binding sites is a positive cis-acting element of its own gene promoter and bip gene promoter in both vegetative and encysting cells. In addition, we found that an ER stress inducer DTT increased both the mRNA levels and promoter activity of the wrky gene. Similar to the results obtained by other lab member, I also found that DTT increased the bip promoter activity. Interestingly, addition of DTT resulted in a change of location of WRKY protein from cytosol to nuclei. Our results suggest that WRKY may be a transcriptional activator for bip gene expression. We also found that WRKY protein can bind to the aTTCAa sequence of the cyclin b gene promoter and this target sequence is similar to the known WRKY binding sequence (g/aGTCAg/a). Overexpression of WRKY protein resulted in an increase of cyclin b gene mRNA, suggesting that WRKY protein may be a transcription activator for cyclin b gene. Our results suggest that WRKY may be an important transcriptional activator and that it may participate in transcriptional regulation of many Giardia genes.