大岩桐兩側對稱花中受CYCLOIDEA調控之轉錄因子

Transcriptional Factors Responsive to CYCLOIDEA in zygomorphic flower of Sinningia speciosa

王佩琦(Jocelin Muliawan)

指導教授 ： 王俊能

國立臺灣大學/生命科學院/生命科學系/碩士(2019年)

https://doi.org/10.6342/NTU201901184

摘要

兩側對稱性花被認定是被子植物演化的主要趨勢，其花從正面可畫出單一個對稱軸，將花分成兩個鏡像半部，背側，兩側和腹側花瓣沿著此對稱軸排列。兩側對稱花使傳粉者從固定的角度進入花中，以促進精確的花粉傳播和柱頭接收，從而大大提高繁殖成功率。在金魚草中，TCP轉錄因子CYCLOIDEA（CYC）在侷限在背部花瓣上表現，CYC透過調節細胞增殖和細胞延長的作用，促使背側花瓣發育，使其在外型上與兩側及腹側花瓣相異。然而，CYC啟動了那些下游基因，以及它們如何合作以產生背部辨識的花瓣形狀和大小是未知的。野生型大岩桐(Sinningia speciosa)為兩側對稱花朵，然而在人為栽培的大岩桐中，兩側對稱卻可輕易地轉換成輻射對稱，這說明了花對稱的發育模組可能是很容易改變。
為了找出CYC可能的下游基因，我們從大岩桐 'Espirito Santo'（SsES）的轉錄組(RNA-seq)中篩選出背腹側瓣之間的差異性表達的轉錄因子（DE-TFs）。其中，篩出9個背側高表達的轉錄因子(包括SsCYC)，其5端調節區(regulatory region)都有鑑定出TCP結合位點，同時也透過qRT-PCR再次驗證這9個轉錄因子確實侷限在背側花瓣表現，因此，這9個轉錄因子很有可能就是SsCYC的下游基因。為了證明SsCYC對這九個轉錄因子的調節能力，在煙草(Nicotiana benthamiana)原生質體的暫時性表達系統中，以雙熒光素酶測定檢測SsCYC和報告子（候選TF的5端調節區）之間的相互作用。結果發現，SsCYC能夠自我調節，並且活化RADIALIS-like（SsRL2）基因，該基因是金魚草中RADIALIS的直系同源基因，但其功能尚不清楚。有趣的是，SsCYC還活化乙烯反應轉錄激活因子SsERF1並抑制乙烯反應轉錄抑制因子SsERF3和ovate家族轉錄抑制因子SsOFP6，其功能目前也尚未知。
SsERF1和SsERF3的可以調控乙烯信號傳導途徑的下游基因。它們可能透過調控EXPANXIN（EXPA）基因、木葡聚醣內轉葡糖基酶/水解酶（xyloglucan endotransglucosylase/hydrolase）基因和內切-1,4-β-D-葡聚醣酶（EGase）基因來使細胞壁變的鬆散，進而改變背側花瓣細胞的延長。同時，這三個基因也在大岩桐轉錄組中被鑑定為背側表達基因，這也符合我們在大岩桐中觀察到背側花瓣的細胞有較大的細胞面積，因此背側花瓣相較於腹側花瓣長度較長，這也被認為是大岩桐花發育成兩側對稱的原因之一。

關鍵字

大岩桐 ； 兩側對稱性 ； SsCYC ； 5端調節區 ； TCP結合位點 ； 下游轉錄因子 ； 背側花瓣 ； RAD ； 乙烯轉錄因子 ； 賀爾蒙 ； 細胞延長

並列摘要

Floral zygomorphy (bilateral symmetry), in which the dorsal, lateral and ventral petals are arranged along a single plane, dividing flower into two mirror-image halves, has been selected as the major trend in angiosperm evolution. Zygomorphic flowers allow the pollinators to enter the flower in fixed angle to facilitate exact pollen deposition and stigma reception, thus greatly enhance reproductive success. In Antirrhinum, TCP transcription factor, CYCLOIDEA (CYC) is strictly expressed at the dorsal petals and it can function to regulate cell proliferation and expansion for generating dorsal identity. However, what the downstream of CYC are and how they cooperate to generate the petal shape and size for the dorsal identity are largely unknown. The wild type Sinningia speciosa exhibits zygomorphic symmetry, yet reversal to actinomorphic (radial symmetry) is common, indicating that the developmental module for floral zygomorphy might be easily altered.
In order to discover CYC downstream, differentially expressed transcription factors (DE-TFs) between dorsi-ventral petals were screened from the RNA-seq data of S. speciosa ‘Espirito Santo’ (SsES). Among them, nine TFs, including SsCYC itself, have their 5’ regulatory regions been identified with TCP binding sites and their dorsal restricted expression was confirmed by qRT-PCR. To demonstrate the possible regulation of SsCYC on these TFs, dual-luciferase assay transiently expressed in protoplasts of Nicotiana benthamiana leaves was used to examine the interaction between the effector (SsCYC) and the reporter (5’ regulatory region of the candidate TFs). It was found that SsCYC was able to auto-regulate itself and also upregulate a RADIALIS-like (SsRL2) gene which is the orthologue of RADIALIS in Antirrhinum, but its function is unknown. Interestingly, SsCYC also up-regulated the ethylene response transcriptional activator, SsERF1 and down-regulated the ethylene response transcriptional repressor, SsERF3 and an ovate family transcriptional repressor, SsOFP6 whose function is unknown.
The finding of SsERF1 and SsERF3 as SsCYC responsive TFs could be linked to their function as downstream regulators of ethylene signaling pathway. They might alter dorsal cell expansion via regulation of EXPANXIN (EXPA) genes, xyloglucan endotransglucosylase/hydrolase (XTH) encoding gene and endo-1,4-β-D-glucanase (EGase) encoding gene to loosen the cell wall, since these three genes were identified as the dorsal expressed genes in the RNA-seq data of SsES. This suggestion is also reflected by the observation that the dorsal petals of SsES have larger cell area, thus are longer in length compared to the ventral petals, which is considered as one of the factors that generates floral zygomophy in this flower.

並列關鍵字

Sinningia speciosa ； floral zygomorphy ； SsCYC ； 5’ regulatory region ； TCP binding sites ； downstream TFs ； dorsal petal ； RAD ； ERFs ； hormone ； cell expansion

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