改良楊樹木質部原生質體轉殖系統以探討被子植物木材形成之轉錄調控機制

Abstract

木材對植物的生長發育扮演重要角色，同時也是人類生活中不可或缺的生物資源。而木材形成是相當複雜的生長發育過程，至今其調控機制所知甚少，且多為單一物種中與少數轉錄因子之研究。因此為系統性地探討木材形成的轉錄調控機制，實驗室先前的研究以木本模式植物毛果楊(Populus trichocarpa)為對象，利用轉錄組測序分析(RNA-sequencing)篩選出182個木質部專一表達之轉錄因子，再加上4個以酵母菌單雜合系統(yeast one-hybrid assay)分析得知會結合木質素生合成基因啟動子的轉錄因子，欲透過DNA親合純化測序分析(DNA affinity purification sequencing)建立木材形成之基因調控網絡(gene regulatory network)。然而該網絡僅能探討轉錄因子能否結合下游基因，無法得知轉錄因子如何進行調控。因此本研究將轉錄因子對毛果楊木質部原生質體進行轉殖以過量表達，並抽取total RNA進行轉錄組測序、篩選差異表達基因，該實驗方法稱為原生質體轉錄體活性分析(protoplast transactivation assay-RNA sequencing)。本研究建立毛果楊木質部原生質體轉錄體活性分析系統，得以大規模對基因調控網絡進行功能性驗證，與DNA親合純化測序分析之結果進行合併便可建構出更加完整的木材形成基因調控網絡。本研究進而對玫瑰桉(Eucalyptus grandis)與中國鵝掌楸(Liriodendron chinense)利用相似策略進行探討，以研究木材形成的調控機制在不同物種中是否存在保守性。透過建立毛果楊、玫瑰桉與中國鵝掌楸轉錄因子家族的親緣關係樹，可得知有若干木質部專一表達的轉錄因子有群聚之現象，顯示這些轉錄因子可能具有相似的調控功能。最後，本研究篩選玫瑰桉中13個木質部專一表達的轉錄因子，以進行往後DNA親合純化測序分析的實驗。未來期望透過比較多個物種之木材形成基因調控網絡，能夠較全面且系統性地探究木材形成的保守轉錄調控機制，以進一步了解木本植物的生長發育過程，並協助木材的育種以及品質改良。

Wood plays an essential role in plant growth, and it is also a valuable bio-based material. Wood formation is a complicated developmental process, but the entire regulatory mechanism is still elusive. Previous studies related to wood formation usually focused on few transcription factor (TF) in single species, thus lacking a comprehensive view of how wood formation is regulated. To obtain more comprehensive knowledge, 186 TFs from Populus trichocarpa were chosen to construct a gene regulatory network (GRN) using DNA affinity purification sequencing (DAP-seq) in our laboratory. Among 186 TFs, 182 TFs were selected as xylem specific TFs by RNA-sequencing, and 4 TFs which physically bind to promoters of lignin biosynthesis genes were selected by yeast one-hybrid assay. However, the network only shows the physical interaction between the TFs and their downstream targets. In this study, the TFs of interested were transiently over-expressed in P. trichocarpa xylem protoplasts. The total RNA was isolated, and carried on to RNA-sequencing. The method was called protoplast transactivation assay-RNA sequencing (PTA-seq). PTA-seq can help us accomplish TF functional characterization in a large scale. Therefore, the combination of PTA-seq and DAP-seq provides a more precise insight in studying wood formation. Similar pipeline was also used to investigate conserved wood formation regulatory mechanism in Eucalyptus grandis and Liriodendron chinense. The phylogenetic trees indicated that several xylem specific TFs are conserved in P. trichocarpa, E. grandis, and L. chinense. Finally, 13 xylem specific TFs from E. grandis were selected for further DAP-seq investigation. We aim to discover the conserved wood formation transcriptional regulatory mechanism in a systematic way, to further support forest tree breeding and wood property improvement.