- 學位論文
以酵母菌減數分裂單雜合及雙雜合系統偵測毛果楊中參與木材形成的轉錄調控複合體
A meiosis-directed yeast one- and two-hybrid coupled system for identifying the transcription factor complexes in wood formation in Populus trichocarpa
摘要
轉錄因子形成蛋白質複合體和DNA產生交互作用，進而影響下游基因的表達，為了要研究生物體的轉錄調控複合體如何影響生物體生長及發育，能透過解析TF-DNA以及TF-TF之間的交互作用，利用酵母菌單雜合(Y1H)以及酵母菌雙雜合(Y2H)系統分別能大規模篩選TF-DNA及TF-TF的交互作用。傳統以Y1H偵測TF-DNA交互作用有其待改進之缺點，包括低TF-DNA交互作用發現率以及耗費時間之操作流程，因此我們建立一個高通量減數分裂法酵母菌單雜合系統(m-Y1H)其具有相較於傳統方法高於雙倍的TF-DNA發現率、較省時之操作流程以及較高的植物體真實TF-DNA交互作用陽性結合率。在過去Y1H與Y2H為了篩選TF-DNA或TF-TF間的交互作用，透過不同的酵母菌菌株帶有不同的報導基因，因此在菌株之差異使Y1H與Y2H不能共用相同酵母菌含有轉錄因子之載體庫，而建立酵母菌含有轉錄因子載體庫需要花上相當多時間及花費，因此我們透過建立酵母菌株Y2HGold-HNG，使減數分裂法酵母菌雙雜合系統(m-Y2H)與減數分裂法單雜合系統(m-Y1H)共同整合，將此新系統命名為減數分裂法酵母菌單-/雙-共雜合系統(m-Y1HY2H)，此系統可以透過相同的酵母菌含有TF-AD庫篩選TF-DNA或TF-TF間的交互作用，除了共用酵母菌株含有TF-AD¬庫的便利性，m-Y1HY2H系統為首創以單倍體與雙倍體酵母菌株細胞篩選TF-TF或TF-DNA間的交互作用，單雙倍體酵母菌之篩選能提供較高的交互作用發現率，發現更多可能在植物體中的交互作用。建立m-Y1HY2H提供一個透過單雙倍體酵母菌細胞篩選TF-DNA或TF-TF交互作用的篩選方式，且為操作時間較為節省時間且容易操作的篩選系統。
並列摘要
Transcription factors (TF) form protein complexes to regulate their targets genes through direct TF-DNA interactions. To identify the TF complexes involved in organism growth and development, two tasks need to be accomplished: the identification of TF-DNA and TF-TF interactions. Yeast one- and two-hybrid systems (Y1H and Y2H) are used extensively to discover TF-DNA and TF-TF interactions, respectively. For TF-DNA interactions, traditional Y1H methods suffer either low discovery rate or time-consuming procedure. We first established a high-throughput meiosis-directed yeast one-hybrid system(m-Y1H), which provides more than 2 times higher discovery rate with short processing time and high in vivo positive rate. In the past, Y1H and Y2H use different yeast strains with their corresponding selection marker for the selection of interactions. In other words, Y1H and Y2H screening cannot share same sets of yeast libraries containing TF vectors, and the preparation of the yeast libraries is dramatically labor- and time-consuming. Here, we constructed a yeast strain, Y2HGold-HNG, which allows us to integrate a new meiosis-directed Y2H system(m-Y2H) onto the m-Y1H. We named this novel system as meiosis-directed yeast one- and two-hybrid coupled system (m-Y1HY2H). In addition to its convenient use, our m-Y1HY2H system can screen the TF-DNA and TF-TF interactions using both haploid and diploid yeast cells, which could not be achieved in previous studies. The double ploidy screening provides higher interaction discovery rates and reveals more potential interaction happened in vivo. This system provides a platform to screen TF-DNA and TF-TF interactions in two kinds of yeast ploidy using the same yeast libraries in a time-saving and easy-to-use way.