- 學位論文
質譜儀對酵母菌基因間隔區的功能探討
Functional Analysis of Intergenic Region in Saccharomyces cerevisiae with Mass Spectrometry
摘要
基因間隔區（Intergenic region, 簡稱IGR）是指位在基因與基因之間的DNA序列，此段DNA序列是屬於非編碼（Non-coding）的，並不會產生蛋白質，因此，基因間隔區在目前認知中被歸類為垃圾DNA（Junk DNA）；然而，在人體基因體結構中約有90%的DNA屬於基因間隔區，佔據了人體基因組的絕大部分，相信基因間隔區似乎有其存在的原因，本篇論文的重點即是想探討基因間隔區域是否有功能以及其角色。 在本論文研究中，以酵母菌為模式生物，主要策略是剔除野生型酵母菌（Wild- type yeast）之基因間隔區，我們稱已剔除基因間隔區之酵母菌為：剔除型酵母菌（IGR-deleted yeast），爾後利用質譜儀分析剔除型酵母菌之蛋白質體，並將其與野生型酵母菌之蛋白質體進行比較分析，比較兩者有無顯著變化的蛋白質。 在本研究中，先挑選出資料庫中尚未被研究且亦沒有顯示出有轉譯RNA的基因間隔區，利用Homologous recombination的方法將設計過後的抗藥性基因（kanMXr gene）置入欲剔除的IGR位置，如此一來除了成功剔除IGR外，還可藉由抗藥性特色篩選出成功剔除的酵母菌。然而，為避免留下的kanMX gene對之後的結果分析產生疑慮，我們利用cre-loxP system，轉入phleomycin-resistent Cre-expressing vector（pSH-ble），將kanMX gene除去而真正的完成了基因間隔區序列的剔除。 同時，利用奈升級液相層析-電噴灑串聯質譜儀分析蛋白質體的改變；實驗的結果發現，利用野生型酵母菌為樣品剔除掉目標基因間隔區的剔除型酵母菌，在質譜分析數據中確實可以找到有特定蛋白質的顯著改變，也成功地利用質譜儀證明基因間隔區在蛋白質的表現上扮演重要的角色；對於發現有所改變的特定蛋白質，在基因的轉譯及轉錄中，是否在pathway上有專一性的影響，也是我們目前積極探討的方向。
並列摘要
An intergenic region (IGR) is a stretch of DNA sequence located between genes. Intergenic regions are non-coding sequences, which cannot be translated to produce proteins. Therefore, in the current cognition, the intergenic region is the so-called “junk DNA”. However, about 90% of human genome sequences belong to the intergenic region. We believe the intergenic region apparently has its reason to exist. The focus of this thesis is to investigate whether intergenic regions have functions or not. In this thesis, we take Saccharomyces cerevisiae (yeast) as the model organism. The main strategy is to remove specific intergenic region of the wild-type yeast with molecular biological methods, and then use the mass spectrometry to analyze its proteomes. We call the yeast strain whose intergenic region is removed the IGR-deleted yeast strain. We compare the proteome of the deletion-type strain with the wild-type strain, and discover that some proteins have significantly changed on expression level. In this study, at first we select the intergenic regions which have not been studied and showed no transcribed RNA in the database. We use homologous recombination method to place the designed anti-G418 gene (kanMX gene) into the position of intergenic region which we would like to remove. As a result, we not only successfully remove the intergenic region, but also screen the successfully IGR-deleted yeast strains according to drug-resistant characteristics. However, in order to avoid the remaining kanMX gene causing unnecessary misgivings in the later analysis results, we utilize the Cre-loxP system by transforming the phleomycin-resistent Cre-expressing vector (pSH-ble vector). With that procedure, we can remove kanMX gene and obtain the yeast strain whose intergenic region is completely removed. At the same time, we use nano liquid chromatography- tandem mass spectrometer (nanoLC-MS/MS) to analyze the proteomes of two type strains, including wild-type and deletion-type yeast. Experimental results show that we can find significant changes of specific proteins. Furthermore, we have also successfully proved intergenic regions play an important role in the expression of proteins by using mass spectrometry. The most important issue we discuss currently is about whether the significant changes of specific proteins related to some biological pathways.