利用基因剔除解析白色念珠菌CaCDC4為非必要基因且扮演型態形成的角色

白色念珠菌(Candida albicans)，是一種伺機性感染的真菌，通常會造成黏膜組織的感染並且嚴重時可造成致命的念珠菌血症， 而其型態間的轉換包括芽胞型態、菌絲型態以及假菌絲型態被認為與其致病能力有很重要的關聯， 但是儘管它跟人類的疾病具有重要相關，對於白色念珠菌的了解卻也未能完全掌握，而其中原因則與它本身的生物特性有關，除了它在自然界通常以雙倍體的形式存在以及沒有一個完整的有性生殖世代交替之外，白色念珠菌在遺傳密碼子的使用上的特殊特性等等的因素，都造成了對其的研究上以及遺傳操作上的一些阻礙與困難。白色念珠菌CDC4基因產物與出芽酵母菌(Saccharomy cescerevisiae)中的CDC4蛋白質具結構同源性，而出芽酵母菌CDC4編碼一種F-box蛋白質是屬於Skp1-Cullin1/Cdc53-F-box(SCF)E3泛素接合酶複合體的成員，其功能為細胞週期中G1-S轉換所必須且在演化上高度保留， 而在本實驗室先前的研究中，對於白色念珠菌CDC4 (CaCDC4)這個在出芽酵母菌中CDC4(ScCDC4)的同源基因，建立了一個可以利用MET3啟動子來加以調控其表現的白色念珠菌品系， 並且意外的發現在抑制MET3啟動子的情況下此品系仍能生長， 而且出現菌絲生長的外表型。為了進一步確認這個結果，在本論文中採用CAURA3 cassette 的方式來建構出兩條CaCDC4對偶基因皆被完整剔除(knockout) 的白色念珠菌品系， 研究結果發現此品系可以生長， 並且出現了菌絲生長的情形，也證明了CaCDC4對於白色念珠菌而言是非必要基因(nonessential gene)，此外，在本實驗中也發現到了CaCDC4 +/- URA 以及CaCDC4 +/- dpl兩種菌株品系在菌絲生長上有程度的差別，推測可能CaURA3也具有影響菌絲生長的可能性。

Candida albicans is known as an opportunistic fungal pathogenthat causes both mucosal infections and life-threatening systemic infections in human. The ability to switch from yeast to hyphal or pseudohyphal form is critical for this multimorphic fungus to invade the host and cause disease.Despite the importance of C.albicans to human disease, the biology and pathogenesis of C.albicans are still illusive, because of its unusual codon usage and an obligate diploid nature lacking a known complete sexual cycle. Candida albicans CDC4 (CaCDC4) encodes a structural homologue of the budding yeast Saccharomyce scerevisiae CDC4 protein product, a F-box protein that is a compoment of the Skp1-Cullin1/Cdc53 -F-box(SCF) ubiquitin E3 ligase, whose function is essential for G1-S transition of the cell cycle that is conserved through out evolution. In our previous study, we have established a strain of C.albicans, which contains one CaCDC4 allele disrupted and the other under MET3 promoter control that is methionine/cycteine repressible, Surprisingly, we have found that under repressed condition this strain is able to proliferate and appears as filamentous phenotype. In order to confirm this discovery, In this study, I use a recyclable CaURA3 cassette to construct a CaCDC4 homozygous null mutant, CaCDC4 -/-, and to examine its phenotypic consequence with CaCDC4 heterozygous null mutants. The results showed that CaCDC4 -/- strain was viable and displays filamentous growth, confirming that CaCDC4 is a nonessential gene for C.albicans. Besides,there were detectable differences between two heterozygous strains CaCDC4 +/- URA and CaCDC4 +/- dpl with the CaCDC4 +/- URA strain having filamentous potential, suggesting that presence of CaURA3 promotes C.albicans filamentous growth.