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標題: | 大岩桐TCP轉錄因子之特性分析 Identification, expression profiles and characterization of the TCP genes in Sinningia speciosa |
作者: | Bo-Hong Yeh 葉柏宏 |
指導教授: | 王俊能(Chun-Neng Wang) |
關鍵字: | TCP轉錄因子,大岩桐,植物發育,基因表現模式,蛋白質交互作用, TCP transcription factor,Sinningia speciosa,plant development,gene expression profiles,protein-protein interaction, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | TEOSINTE-BRANCHED 1/CYCLOIDEA/PCF (TCP)家族為植物獨有的轉錄因子,到了開花植物,由於基因不斷地複製特化產生新功能,其成員廣泛地被報導參與調控植物形態生長、營養及生殖器官發育,包含枝條、葉、花部、尤其是花兩側對稱性發育。然而,過往僅有少數非模式物種之 TCP轉錄因子基因家族被全面地進行序列釣取及檢驗其表現,以了解其參與了哪些器官的重要發育。野生種大岩桐有兩側對稱花,及突變反轉成輻射對稱花的馴化栽培品系;而且大岩桐屬植物,其葉部形態具有豐富的變異,可用來檢驗其TCP家族成員是否參與了花或葉的發育過程。為了解TCP基因在大岩桐的角色,我們從花瓣轉錄體中鑑定出30個TCP基因。為了釐清TCP基因對花及葉的發育角色,我們先重建大岩桐所有TCP基因演化樹,結果發現有15個Class I TCP基因和15個Class II TCP基因,其中Class II 包含3個CYC/TB1亞群TCP基因以及12個CIN亞群TCP基因。進一步分析TCP基因的序列結構,結果發現在CYC/TB1亞群中的TCP基因(SsTCP1, 13, 22)皆具有R domain,推測與蛋白質之間的交互作用有關。另外在CIN亞群中,SsTCP 19, 20, 27, 28, 29具有被miR319a調節的辨識位,可能和之前文獻報導的葉上下表面極性發育有關。而在RT-PCR檢驗其在發育時期及組織表現位置的結果,發現Class I TCP基因廣泛地在營養時期和繁殖時期有表現,Class II的CYC/TB1亞群主要在花表現,而在CIN亞群主要在葉表現。且大岩桐TCP基因數量比阿拉伯芥多,顯示似乎有多次基因複製多樣化現象,且這些複製出的基因有表現形式互補的現象或是組織專一性情況。如 CYC/TB1亞群中,SsTCP1, 22在花的時期有高表現,而CIN亞群的TCP基因在營養時期的組織有高表現。另外,SsTCP9、SsTCP22只在繁殖時期有表現以及背側花瓣的表現量高於腹側花瓣。另一方面,先前研究指出TCP轉錄因子常需透過形成同型或異型雙聚合體之方式對下游基因進行調控,且有些 TCP轉錄因子會進行自體調控或是相互調控。本研究以酵母菌雙雜合系統,來檢驗TCP蛋白間的交互作用關係,進一步推測其如何參與大岩桐的發育。結果顯示SsTCP有7個的同型蛋白質雙聚合體和57個異型蛋白質雙聚合體產生。而CIN亞群和CYC/TB1亞群相較下,CIN亞群基因間幾乎兩兩都有交互作用,而在CYC/TB1亞群基因間,則少數具有同型雙聚合體或異型雙聚合體產生,且與CIN亞群之間的蛋白質交互作用也較少。表示TCP轉錄因子傾向於各自分群內形成同體或異體雙聚合體,而群間有較少的交互作用。另外,CIN亞群在營養器官中會廣泛表現,可能是因傾向和其他TCP蛋白質形成雙聚合體,來一同調控下游基因。因此透過全面性的分析大岩桐TCP家族成員有助於進一步的瞭解其演化關係及基因特性,替往後研究,如功能性分析提供了TCP基因之基礎訊息。 The TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) family is a group of plant-specific transcription factors which obtain new functions after gene duplication in angiosperm. TCP family involves in wide range of plant developmental pathways such as branching, leaf development, floral organ morphogenesis, and especially in flower bilateral symmetry regulation. However, a little information about comprehensive analysis in roles of TCP family in non-model organisms, including identification of TCP genes and analysis of expression profile. Wild-type S. speciosa flower is bilateral symmetry, cultivated flower is radial symmetry and abundant variation in leaf shape. The TCP genes may modulate in development of flower or leaf. To explore the roles of TCP proteins on flower or leaf development of S. speciosa, a total of 30 TCP genes (SsTCPs) were identified from flower transcriptome of S. speciosa. The homology of these SsTCPs were clustered into class I and class II based on the sequence similarities in phylogeny. The class II SsTCPs can be further subdivided into subfamily of TB1/CYC and CIN. The SsTB1/CYC subclass homologs we found all contained R domain that presumably mediate protein–protein interaction. Those in the CIN subclass have mir319a targeting site implying they can be regulated for leaf morphogenesis. Additionally, expression profiles of these class I and class II SsTCPs were examined in different flower stages and vegetative organs to clarify their possible roles. Most of class I TCP genes were widely expressed through vegetative phase and reproductive phase. The CYC/TB1-type genes were highly detected in flowers, while the CIN-type genes were highly detected in leaves. In addition, the number of TCP genes in S. speciosa was higher than that in Arabidopsis. It is suggesting that the expansion of TCP family in S. speciosa may be caused by gene duplication, and these duplicate genes exhibited complementary or tissue-specific patterns. For example, in TB1/CYC subclass, SsTCP 1 and 22 were highly expressed in flower. In CIN subclass, SsTCP genes were highly expressed in vegetative phase. SsTCP 9 and SsTCP 22 only expressed in flower organs and highly expressed in dorsal petal than ventral petal. On the other hand, TCP proteins tend to form homodimers or heterodimers with other TCP proteins, and dimerization may be required for their DNA-binding activity and hence for their biological activity. Therefore, we performed yeast two-hybrid assays to examine dimer formation among class II SsTCP proteins. The results showed the 7 homodimer formations and 57 heterodimer formations, and CIN subfamily have more abundant interactions than CYC/TB1 subfamily. It suggested TCP proteins form homodimers and heterodimers particularly with proteins within the same class. Furthermore, SsTCP proteins of CIN subfamily tend to form dimer with other TCP proteins, and widely expressed in vegetative organs to modulate the downstream genes. Together, these results shall clarify possible evolutionary relationships of TCP family, and may provide the foundation for further study on the functions of TCP genes in S. speciosa development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7633 |
DOI: | 10.6342/NTU201800221 |
全文授權: | 同意授權(全球公開) |
電子全文公開日期: | 2028-12-31 |
顯示於系所單位: | 生命科學系 |
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