二、 材料
5.10 REP5 基因不會影響白色念珠菌之型態轉換，且 REP5 基因過度表現株不會影
REP5基因位於白色念珠菌contig19-10215中，ORF號碼為19.4884。Rep5p具有785個 胺基酸，大小約86.35 kDa。經過序列比對分析後發現，REP5基因和啤酒酵母的ORF YEL007w具有相似性。YEL007w為具有666個胺基酸的蛋白質，於啤酒酵母中的功能未 知。Rep5p的6 ~ 792個胺基酸與YEL007w的3 ~ 660個胺基酸之間的相同性(identity)達27
%，相似性(similarity)達40 %(Evalue= 5e-27)(圖二十五)。又REP5基因和YEL007w皆與 Schizosaccharomyces pombe中與gluconate transport induction有關的Gti1p具有微弱之相似 性(但N端前80幾個胺基酸相同性高達40 %以上，且此範圍內包含了一個可能的Pka1 phosphorylation site KRWTDG)(圖二十五)(Caspari, 1997)。先前研究發現，白色念珠菌的 a和α細胞必須先經過表型轉換(phenotypic switch)，從white階段轉變成opaque階段才有交 配(mating)能力(Miller & Johnson, 2002)。研究發現，在白色念珠菌中，REP5(YEL007w) 基因會在a和α細胞內表現，但不會在a/α細胞表現，故REP5(YEL007w)基因的表現會受 a1/α2調控(被抑制)(Tsong et al., 2003)。又，Micheal等人的研究發現，在HBR1/hbr1(HBR1 為白色念珠菌white-opaque轉換的抑制者)異型缺陷突變株內與MTLa1與MTLα1,2的缺陷 株內，REP5(YEL007w)基因的mRNA含量有提高現象(約提高2 ~ 3倍)(Pendrak et al., 2004)。先前研究得知，在啤酒酵母2B/int5314CDR1p-lacZ 中，REP5基因能夠提高 β-galactosidase 活性約7倍(紀錦昇，交大碩士論文，2004)。又，實驗發現REP5基因能夠 活化FLO11::lacZ的表現(Rottmann et al., 2003)，而Flo11p/Muc1p已知為啤酒酵母中和 pseudohyphal以及invasive growth有關的蛋白質(Lambrechts et al., 1996; Lo & Dranginis, 1998)。綜合上述結果，為了想知道REP5基因是否牽涉到抗藥性和型態(morphology)變 化，便開始著手研究REP5基因。
由於一直無法順利得到rep5/rep5同型缺陷突變株，故採用四環黴素調控系統 (Nakayama, et al., 2000)並在有無添加doxycycline的情況下，調控REP5基因的表現形式。
添加doxycycline時，REP5基因會受到抑制而不表現；不加doxycycline時，REP5基因會 過度表現。由於doxycycline會影響野生型SC5314生長狀況，使SC5314菌株生長受到抑 制，菌落變得較衰弱。為了避免結果難以判讀與解釋，故於之後的藥物敏感性實驗，都 不添加doxycycline(不觀察REP5基因抑制現象)，只觀察REP5基因過度表現之影響。由圖 二十二及二十三之結果得知，不論於藥物敏感性試驗(Etest)或是agar dilution assay中，在 不添加doxycycline的狀況下(REP5基因過度表現)和野生型SC5314(REP5/REP5)相比，
REP5基因過度表現株WJC58與WJC63，對於azoles類藥物- fluconazole、 ketoconazole、
itraconazole、voriconazole以及miconazole和polyenes類藥物-amphotericin B 的藥物敏感性 皆無顯著變化。而在型態研究方面得知，在誘導菌絲形成之狀況中(37 ºC高溫培養及加 入10 %胎牛血清)，加入doxycycline抑制REP5基因表現時，仍然會有芽管生成(圖二十 四)。綜合所有實驗結果發現：REP5基因不會影響白色念珠菌之型態轉換，且REP5基因 過度表現株WJC58與WJC63不會影響白色念珠菌之藥物敏感性。為了更清楚得知REP5 基因是否和藥物敏感性有關，我們必須再次利用同源重組置換方式完整破壞REP5基 因，希望能夠得到rep5/rep5同型缺陷突變株，以供近一步研究REP5基因和抗藥性之間的 關係。
六、未來展望
1. 已知rep3/rep3同型缺陷突變株對於azoles類藥物fluconazole、voriconazle和miconazole 的敏感性提高，變得較敏感。此現象可於rep3/rep3::REP3單套基因置入補救株中驗 證。由於本論文之agar dilution assay研究只有觀察菌株對於miconazole的敏感性現 象，故之後可以fluconazole及voriconazole進行agar dilution assay，觀察是否同樣具有 和藥物敏感性試驗(Etest)相同之結果。
2. 探討野生型SC5314(REP3/REP3)、rep3/rep3同型缺陷突變株與rep3/rep3::REP3單套 基因置入補救株中排藥幫浦CDR1 mRNA含量。如果REP3基因藉由直接和CDR1作用 而造成白色念珠菌之抗藥性生成，那麼我們應該可在rep3/rep3同型缺陷突變株內觀 察到CDR1 mRNA表現量下降，而於rep3/rep3::REP3單套基因置入補救株中，CDR1 mRNA表現量會回升。
3. 以四環黴素調控系統過量表現REP3基因，觀察REP3基因過量表現時的藥物敏感性 現象是否會與rep3/rep3同型缺陷突變株相反。
4. 再次利用同源重組置換實驗破壞REP4基因，並以所得到的rep4/rep4同型缺陷突變株 驗證藥物敏感性實驗(Etest)中的正確表型，之後可於實驗中觀察rep4/rep4同型缺陷 突變株、rep4/rep4::REP4單套基因置入補救株與rep4/TR::REP4基因過度表現株中的 排藥幫浦CDR1 mRNA表現量，藉此研究REP4基因和CDR1之間的作用。
5. 利用同源重組置換實驗完整破壞 REP5 基因，並以所得到的 rep5/rep5 同型缺陷突變 株進行藥物敏感性實驗(Etest)，試著找出 REP5 基因和 CDR1 之間的相關性。
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圖一. rep3/rep3::REP3 單套基因置入株建構示意圖。
A/U: 表示 ARG4 或 URA3 篩選標誌。A＇表示 open reading frame 不完整的 A 基因; B＇表示 open reading frame 不完整的 B 基因。
HIS1 A’ B
A/U B
HIS1
pGEM-HIS1
A’
B’ REP3
A/U A
A
REP3 B＇
homologous recombination at AflⅡ site
AflⅡ
AflⅡ
A＇
2.6 kb
B
3.7 kb or 3.4 kb
A B’ HIS1 REP3 B
HJL458
HJL458 HJL505
HJL505
A/U
A/U A’
rep3/rep3 allele (A)
(B)
圖二. 聚合酶連鎖反應確認 REP3 單套基因是否正確置入 rep3/rep3 同型缺陷突變株內。
圖二. 聚合酶連鎖反應確認 REP3 單套基因是否正確置入 rep3/rep3 同型缺陷突變株內。