使用減數分裂－酵母單雜交方法研究毛果楊中木材形成的基因調控網路

Abstract

基因調控網路 (gene regulatory networks, GRNs) 可以幫助人們瞭解在基因進行轉錄表達的時候調控因子 (transcription factor, TF) 和目標基因 (target gene) 之間的相互作用關係，而酵母單雜交 (yeast one-hybrid, Y1H) 系統是可以快速建立這種網路的研究方法之一。酵母單雜交的原理是分別將含有目標基因接著報導基因的質體和含有TF表達序列的質體轉殖到酵母菌中，使TF和目標DNA存在於同一個細胞裡，然後通過對這種酵母菌細胞中報導基因表達的篩選，來偵測TF和DNA是否有交互作用。在過去的研究中，酵母單雜交依照操作方式分為兩種類型：單倍體轉殖法 (haploid-transformation) 和雙倍體交配法 (diploid-mating)。 我們透過酵母單雜交系統研究木材合成相關的TF和DNA之間的結合，來初步建立木材形成相關的基因調控網路。本文以木本模式植物毛果楊為研究物種，挑選與木材形成相關的基因為研究材料。接著基於傳統酵母單雜交系統，我們引入了合成遺傳陣列 (synthetic genetic array, SGA) 技術，開發出一種適應高通量操作的新酵母單雜交系統，即減數分裂－酵母單雜交系統 (meiosis-directed Y1H system)。 通過和傳統的酵母單雜交系統進行測試對比，我們發現這種減數分裂－酵母單雜交系統，能夠進行快速簡便的高通量操作，並且得到最高的偵測結合數量，是一種可以快速高效建立基因調控網路的研究方法。

Gene regulatory networks (GRNs) can demonstrate the interactions between transcription factors (TFs) and their target genes, which would be detected by yeast one-hybrid (Y1H). In Y1H, two components are needed: DNA baits and TF preys, and the interactions between TF and their target DNA are measured by the downstream reporter gene activation in yeast. In the past studies, the yeast one-hybrid system was categorized into two different methods: haploid-transformation and mating diploid-mating. In our study, a high-throughput meiosis-directed Y1H system was developed by synthetic genetic array (SGA) technique. After testing meiosis-direct method, haploid-transformation and diploid-mating method together, we found that the novel method has benefits in highest discovery rate and short processing time. This meiosis-direct Y1H system can be used to quickly constructing the gene regulatory network in wood formation.