白色念珠菌同型合子 (Homozygote) 之營養要求菌株之誘導與有性器官之觀察

Mutgenesis of auxotrophic homozygous strains and observation of sexual organ on Candida albicans

鍾欣蓉(Hsin-Jung Chung)

指導教授 ： 蘇慶華

臺北醫學大學/醫學院/醫學科學研究所/碩士(2009年)

https://doi.org/10.6831/TMU.2009.00131

摘要

白色念珠菌被認為是絕對二倍體的酵母菌，其有性器官和減數分裂至目前為止尚未被發現，然而卻發現其可藉由二倍體間的交配完成擬有性世代 (parasexual cycle)。本實驗室先前的研究中指出在Azole類藥物 (Fluconazole)、Terbinafine的壓力刺激下，同型合子 (Homozygote) 的opaque phase以高機率發生於PDA/phloxine B的抑制圈內，並分離出不同性別之同型合子菌株。這暗示著白色念珠菌可能在Fluconazole處理的環境下進行減數分裂。本研究利用二倍體和單倍體染色體的不同，照射紫外線 (UV) 後存活率不同，可初步判別a type菌株的倍數體。此外，也以UV照射和複印法 (Replica) 篩選出同對histidine具營養要求性的單倍體營養要求突變菌株共23株，其中包括a type SW08 14株；a type 4F 7株；α type GB2 2株，經由9種含4~5種不同胺基酸或含氮鹼基的培養基交叉篩選，發現所有的菌株均對histidine具營養要求性。另外，在形態上以DAPI染色由共軛焦顯微鏡發現經由Fluconazole處理並給予麥角固醇 (Ergosterol) 之培養基的抑制圈內，白色念珠菌會產生類似豆莢型 (peapod) 且多核之類子囊結構；含有0.1% Squalene的PDA/Phloxine B培養基中，更可發現包裹類子囊孢子 (ascospore-like) 之成熟類子囊 (ascus-like) 結構。同時，以掃描式電子顯微鏡觀察可發現塌陷、扁平的破裂類子囊。這些形態證據顯示Peapod極有可能是白色念珠菌的有性器官—子囊 (ascus)，但仍需藉由其他遺傳學與分子生物學的證據佐證。

關鍵字

白色念珠菌 ； 營養要求菌株 ； 有性器官 ； 鲛鯊烯 ； 麥角固醇

並列摘要

Candida albicans is considered as an obligate diploid budding yeast and the meiosis and the following formation of sexual organs is not been found however a parasexual cycle to provide genetic variation is confirmed. Our previews study showed antifungal agents such as azoles and terbinafine enhanced a high frequency of homozygosis in the sub-MIC zone on PDA-phloxine B medium and homozygous strains with different mating types were isolated. This result indicated that the possibility of meiosis might exist in fluconazole treated system. In the present study, the homozygous strains of different gender isolated from fluconazole treatment were subjected for mutagenesis by UV radiation for the selection of auxotrophic mutant and the killing curve of each strain was measured in the same time for the determination of ploidy. All the a type strains showed a more susceptible to UV radiation with a steeper curve of survival rate with higher tendency to be haploid strains. For the screening of auxotrophic mutants, replica technique was employed and totally 23 strains were selected in which 14 strains were from SW08 (a type), 7 from 4F (a type) and 2 from GB2 (α type). The nutrient deficiency of each auxotrophic mutant was determined by cross-plating on 9 media containing 4-5 amino acids or nucleotides. The results indicated that all the strains were in histidine requirement. In the meantime, a peapod shape, multi-nuclei, ascus-like cell structure was found in the cloudy zone during fluconazole with ergosterol supplement to C. albicans under DAPI/confocal microscopy. Furthermore, the ascus-like structure was found in 0.1% squalene PDA-phloxine B medium. Collapsed ascus-like cells also found in 0.1% squalene PDA-phloxine B medium under scanning electron microscopy (SEM). It suggested the peapod shaped structure might be the sexual organ of C. albicans. However, more other evidences of genetics and the molecular biology are required to support this hypothesis.

並列關鍵字

Candida albicans ； auxitrophic strain ； sexual organ ； squalene ； ergosterol

參考文獻

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