Abstract
摘要:生物能因應各種環境的變化,調整本身的生理狀態。在自然環境中,光為一重要的環境因子,除了為能量來源,光線的變化也調控自然界中各種生物的生長及發育。在真菌光反應的研究中,Neurospora crassa為主要的研究模式,N. crassa可感受藍光,藍光調控其無性及有性之產孢過程,並且亦為其生物週期重要環境影響因子之一。在N. crassa的研究中得知,wc-1及wc-2基因為N. crassa光反應主要之調控因子,2002年發表於”科學”雜誌期刊之兩篇報導,更進一步被証實WC-1為其藍光的光接受器,此乃真菌第一個被證實之光反應接受器。此外,因真菌基因體研究之快速發展,許多模式真菌光反應之分子機制,亦積極地被研究中。隱球菌(Cryptococcus neoformans),為一伺機性人體病原真菌,已具完備的分子生物學研究系統,加上已完成之基因體計劃,其已發展為真菌致病性、生理及發生學分子調控機制之模式生物。隱球菌有性生殖之費洛蒙調控制之研究中,吾人發現隱球菌有性生殖雙核菌絲之形成與生長,受光線抑制。在本計畫94年度執行期間,吾人已界定隱球菌只可感受可見光譜中之藍光,為進一步探討隱球菌光反應之分子調控機制,吾人於隱球菌中找到N. crassa wc-1及wc-2之同源基因,並分別命名為CWC1及CWC2,利用遺傳工程技術,建構獲得cwc1及cwc2突變株,並證實該二基因確實參與隱球菌光反應之調控。相關研究成果已發表2005年“Molecular Microbiology 56: 480-491” (SCI,Impact factor 6.203)。杜克大學另一實驗室之研究發現,CWC1及CWC2基因亦參與病原性之調控。為進一步探討CWC1及CWC2之藍光分子調控機制,尋找隱球菌Cwc蛋白質之作用蛋白及其下游基因,已利用農桿菌突變技術進行suppressor 篩選,尋找Cwc1及Cwc2之下游基因。在目前所篩選的4,132株農桿菌轉殖株,其中64株具有回復CWC1大量表現株抑制
Abstract: All organisms respond to environmental stimuli and make appropriate physiological adjustments. Light is an important environmental signal and has profound effects on living creatures. Light not only is the major source of energy on Earth, and the fluctuation of light also regulates the growth and differentiation of many organisms. In fungi, Neurospora crassa has been the leading model for studying the molecular mechanisms of blue light photobiology. Blue light regulates asexual and sexual sporulation process, and also acts as a signal to entrain the circadian rhythm. In N. crassa, wc-1 and wc-2 genes are the central regulators of the blue light responses and all light responses are lost in these two mutants. In 2002, two articles published in “Science” demonstrated that WC-1 is the blue light photoreceptor of N. crassa. This is the first photoreceptor identified in fungi. Recent advance in fungal genomic studies have stimulated the investigation of photoresponses in various model fungal system. Cryptococcus neoformans, an important human fungal pathogen with well-established molecular biology systems and five different genomes sequenced, is suitable for studying the molecular mechanisms of fungal pathogenesis, physiology and differentiation. Studying the C. neoformans genes involved in the mating process, we observed that production of the dikaryotic filaments during the mating process is inhibited by light. In studies supported by NSC, we determined that C. neoformans also perceives blue light in the visible light spectrum. To dissect the molecular mechanisms of blue light responses in C. neoformans, we identified the N. crassa wc-1 and wc-2 homologues in the C. neoformans genome and named the genes CWC1 and CWC2 respectively. To determine their roles in C. neoformans, we generated the cwc1 and cwc2 gene deletion mutants. Deletion of both genes resulted strain insensitivity to light and sexual filamentation is no longer inhibited by light. On the other hand, overexpression of both genes under a constitutively expressed promoter inhibited filamentation, and interestingly such inhibition is strictly dependent upon light irradiation. Our investigation demonstrated that blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans. Our work was published in “Molecular Microbiology (2005) 56:480-491” (SCI, Impact factor 6.203). Interestingly, Cwc1 and Cwc2 were also shown to contribute to the virulence of this human fungal pathogen by a research group at Duke University. In continuation of our studies and also reveal how they regulate the virulence, we initiated a suppressor screening and tried to identify the downstream targets of Cwc1 and Cwc2 in the photoregulated pathway. Using Agrobacter
Keyword(s)
隱球菌
CWC1基因
CWC2基因
藍光光反應
分子機制
農桿菌突變技術
suppressor 篩選
Ssn8
Set2
Bud16
Mub1
Cryptococcus neoformans
CWC1
CWC2
blue light photoresponse
molecular mechanism
Agrobacterium-mediated mutagenesis
suppressor screen
Ssn8
Set2,