隱球菌光反應之研究與隱球菌SSN8同源基因之探討

Dissection of the Cryptococcus neoformans light response and characterization of Cryptococcus neoformans SSN8 homologue

林裕盛(Yu-Sheng Lin)

指導教授 ： 沈偉強

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00628

摘要

隱球菌為人體重要病原真菌。實驗室過去的研究發現，藍光抑制隱球菌生殖菌絲的形成。進一步利用同源序列的比對，在隱球菌基因體中，找到真菌保守性藍光調控基因CWC1及CWC2，並證明二者為隱球菌藍光反應不可或缺的因子(Lu et al., 2005)。為進一步探討隱球菌光反應Cwc複合體下游抑制菌絲形成的相關機制，本研究以農桿菌逢機突變轉殖技術，利用 CWC1基因過度表現株完全抑制生殖菌絲形成的特性，進行回復生殖菌絲生長突變株之篩選。利用此suppressor screening的策略，經篩選4132個農桿菌轉殖株，其中64株具有不同程度回復生殖菌絲生長之情形。其中一農桿菌轉殖株AZ5，T-DNA插入破壞CWC2基因之表現，印證了本篩選策略的有效性。另一轉殖株AY18，經實驗證明，T-DNA插入破壞隱球菌SSN8同源基因，並因此造成生殖菌絲回復生長之特徵。啤酒酵母菌SSN8基因，又稱為SRB11/UME3/CYCC/RYE2，為轉錄調控mediator complex的一員，參與糖類利用、減數分裂、細胞生長，以及轉錄反應等生理之調控。為瞭解SSN8基因在隱球菌中所扮演之角色，本研究亦建構野生型ssn8突變株、回復突變株，及過度表現株。SSN8基因突變造成的性狀包括，生殖菌絲的減少， haploid fruiting菌絲的大量生成、黑色素的明顯累積，而在營養充分的培養條件下，ssn8突變株細胞形態發生改變，並且在固體培養基表面可見侵入菌絲的形成等。相關菌株性狀分析的結果顯示，SSN8基因參與隱球菌許多生理及發生的過程，並且為負向調控之角色，而SSN8基因如何調控相關生理反應，以及其與Cwc複合體間的關連性，有待進一步的探討。

關鍵字

隱球菌 ； 藍光反應 ； CWC1 ； CWC2 ； 農桿菌逢機突變篩選 ； SSN8同源基因 ； mediator complex

並列摘要

Cryptococcus neoformans is an important human fungal pathogen. In our previous investigation, we demonstrated that blue light negatively regulates the sexual filamentous growth via the Cwc1/Cwc2 complex in C. neoformans. To further understand how light inhibits mating filamentation, we conducted a suppressor screening using Agrobacterium-mediated insertional mutagenesis to identify the components interacting with or downstream of the Cwc complex under the CWC1 overexpression strain background. Among 4132 nourseothricin-resistant transformans screened, we identified 64 strains restored different levels of mating filamentation. The sites of T-DNA integration among 21 transformans were recovered. One strain, AZ5, restored the highest level of filamentation was observed and that T-DNA was found to be inserted at the 5’-UTR region of the CWC2 gene. The finding of CWC2 gene in the screen validates our approach. In this study, we further characterized the transformant AY18 in which T-DNA integration affected the activity of a gene homologous to the Saccharomyces cerevisiae SSN8. In budding yeast, SSN8 gene, also named SRB11/UME3/CYCC/RYE2, has been demonstrated to play diverse roles in sugar utilization, meiosis, cell phase control, and transcription regulation. To reveal the roles of SSN8 gene in C. neoformans, we generated the ssn8 mtant, reconstituted strain, and overexpression strain under the wild-type background. The mutant phenotypes included slightly reduced mating filamentation, extensive haploid filamentation, increased melanin accumulation, changed cellular morphology and production of invasive hyphae under rich YPD growth condition. Our results suggest that C. neoformans SSN8 homologue plays critical roles in the diverse physiological and differentiation processes. This is the first demonstration of mediator component in C. neoformans. Its roles related to the Cwc complex remain to be further investigated.

並列關鍵字

Cryptococcus neoformans ； blue light response ； CWC1 ； CWC2 ； Agrobacterium random insertional mutagenesis ； suppressor screen ； SSN8 homologue ； mediator complex.

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