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研究生：	郭玹州 Kuo, Xuan-Zhou
論文名稱：	探討 ABC 轉運蛋白參與大葉蝴蝶蘭單萜類之花香釋放 Investigation of ABC transporters on monoterpene emission for Phalaenopsis bellina floral scent
指導教授：	陳虹樺 Chen, Hong-Hwa
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生命科學系 Department of Life Sciences
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	51
中文關鍵詞：	ABC轉運蛋白、香味釋放、基因靜默、單萜類、PbABCG 、蝴蝶蘭
外文關鍵詞：	ABC transporter, floral scent, gene silencing, monoterpenes, PbABCG, Phalaenopsis
相關次數：	點閱：107 下載：0
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大葉蝴蝶蘭是蝴蝶蘭中帶有香味的原生種，主要香味成分為單萜類(monoterpenoids) 中的linalool、geraniol和其他衍生物。在過去的研究中已知大葉蝴蝶蘭的花香生合成路徑，然而花香從大葉蝴蝶蘭中釋放出來的機制尚不清楚。在矮牽牛花上已經發現ABC轉運蛋白能協助花香釋放。本論文研究進一步評估ABC轉運蛋白是否參與大葉蝴蝶蘭的花香釋放。首先在大葉蝴蝶蘭的轉錄組基因庫中鑑定出98個ABC轉運蛋白相關基因，經過演化樹分析發現其中47個基因聚集在與萜類化合物轉運有關的亞家族 G中。通過與不具香味物種台灣白花蝴蝶蘭進行差異性基因表現分析，進一步再以ContigViews分析，發現 PbABCG1、PbABCG2 和 PbABCG5 與PbGDPS和PbbHLH4呈現基因表現高度相關。再以qRT-PCR得出 PbABCG1、PbABCG2及PbABCG3在開花後第5天，即花香釋放最高時具有出最高的基因表現，而PbABCG4、PbABCG5及PbABCG6 則在開花第一天具最高的基因表現。因此PbABCG1及PbABCG2 較有可能參與在大業蝴蝶蘭之花香釋放。在空間上，PbABCG1和PbABCG2均在根和葉中表達，在花的部分PbABCG1在萼片中表達最高，而PbABCG2則在唇瓣具有較高表現。另外，在亞家族B中也發現一個基因PbABCB1與PbGDPS和PbbHLH4呈現高度相關。但經過qRT-PCR確認後其最高的基因表現在開花後第13天，顯示PbABCB1應無參與在大葉蝴蝶蘭之花香釋放。在基因功能分析上，利用失去功能的方法測試，包括菸草脆裂病毒誘導的基因靜默和RNA干擾技術，分別對PbABCG1和PbABCG2各別兩段位於保守序列Walker A和Walker B下游長度為100-bp的基因進行。結果顯示PbABCG1 及PbABCG2基因表現量在病毒誘導的基因靜默植株PbABCG1-1、PbABCG1-2、PbABCG2-1和PbABCG2-2分別降低基因表現為67%、27%、50%、59%，在單萜釋放量分別降低為74%、30%、19%、40%。在RNA干擾上，PbABCG1-1、PbABCG1-2、PbABCG2-1和PbABCG2-2分別降低PbABCG1及PbABCG2基因表現為46%、30%、67%、47% ，而在單萜釋放量分別降低為32%、21%、46%、5%。且在病毒誘導的基因靜默及RNA干擾實驗中，對PbABCG1基因的下調並不會影響PbABCG2基因的表現，反之亦然，顯示具有基因專一性。此外，PbABCG1和PbABCG2的異位表達也增強了酵母菌對香葉醇的耐受性。總結我的實驗結果證實PbABCG1和PbABCG2對P. bellina花香釋放單萜類有重要作用。

Phalaenopsis bellina is a scented Phalaenopsis species with the main floral scent components of monoterpenes, including linalool, geraniol, and their derivatives. Previously, our lab has identified the floral synthesis pathway of P. bellina. However, the mechanism which the floral scent is released from the P. bellina is unknown. The ABC transporter has been found can assist in the release of floral scent in petunia hybrida. This thesis assessed whether ABC transporters are involved in floral scent emission in P. bellina. First, a total of 98 ABC transporter-related genes were identified in the transcriptomics libraries of P. bellina. Among them, 47 genes were clustered in the subfamily G related to the transport of terpenoids by use phylogenetic analysis. Compared with a scentless species P. aphrodite, three genes were found to be related to scent phenotype, including PbABCG1, PbABCG2 and PbABCG5. Further analysis of gene-to-gene correlation by use ContigViews showed that PbABCG1 and PbABCG2 were highly correlated with PbGDPS and PbbHLH4. Both temporal and spatial gene expression were analyzed by using qPCR. PbABCG1, PbABCG2 and PbABCG3 showed optimal expression on day 5 post-anthesis concomitant to the peak floral scent emission. In contrast, PbABCG4, PbABCG5 and PbABCG6 showed high expression on 1 day post-anthesis (D+1). These suggest only both PbABCG1 and PbABCG2 were involved in floral scent emission. Spatially, both PbABCG1 and PbABCG2 expressed in root and leaf, and PbABCG1 highly expressed in sepals, while PbABCG2 highly expressed in lip. In addition, an ABC transporter subfamily B gene, PbABCB1 showed high correlation with PbGDPS and PbbHLH4. However, its gene expression showed high expression on D+13, suggesting that PbABCB1 was most likely not involved in floral scent emission. To perform functional analysis, loss of function assays were conducted by use of tobacco rattle virus (TRV) - induced gene silencing (VIGS) and hairpin RNA induced (RNAi) gene silencing system. Downregulation of two gene specific 100-bp fragments downstream from the conserved Walker A and Walker B for both PbABCG1 and PbABCG2 individually. This led to the reduced gene expression of PbABCG1 and PbABCG2 to 67%, 27%, 50% and 59%, and the monoterpene emissions were also significantly decreased to 74%, 30%, 19% and 40%, respectively in PbABCG1-1, PbABCG1-2, PbABCG2-1 and PbABCG2-2-silenced plants. In RNA interference, it reduced gene expression of PbABCG1 and PbABCG2 to 46%, 30%, 67% and 47%, and the monoterpene emissions were also significantly decreased to 32%, 21%, 46% and 5% in PbABCG1-1, PbABCG1-2, PbABCG2-1 and PbABCG2-2-RNAi plants. In TRV-based VIGS and RNAi experiment, downregulation of PbABCG1 gene did not affect the performance of PbABCG2 gene, and vice versa, suggesting that the silencing effect showed great gene specificity. In addition, ectopic overexpression of PbABCG1 and PbABCG2 in the ABC16- mutant yeast strain rescued its tolerance to geraniol. Together, my results confirmed that PbABCG1 and PbABCG2 played substantial roles in the transport/emission of monoterpenes for P. bellina floral scent.

Table of content

中文摘要……………………………………………………………………I
ABSTRACT………………………………………………………………………III
誌謝…………………………………………………………………V
List of Table ……………………………………………………………VIII
List of Figure ………………………………………………………………IX
List of Supplementary Figure………………………………………XI
1.	INTRODUCTION……………………………………………………1
1.1 The economic value of orchids in Taiwan………………………………1
1.2 Scent species of Phalaenopsis……………………………………………1
1.3 The floral scent compounds in Phalaenopsis bellina…………1
1.4 The emission mechanisms for floral volatiles…………………………3
1.4.1 The signals for floral scent emission……………………………3
1.4.2 The emission mechanisms for floral volatiles………………3
1.5 The influence and importance of ABC transporters in plants………3
1.5.1 ABC transporters classifications……………………………4
1.5.2 The role of ABC transporters on plant signal transmission………4
1.6 The influence and importance of ABC transporters for plant secondary metabolite 5
1.6.1 Known ABC transporters on emission of plant secondary metabolites …5
1.6.2 The role of ABC transporters on emission of plant volatiles ……5
PURPOSE ………………………………………………………7
2.MATERIAL and METHODS ……………………………8
2.1 Plant materials………………………………………8
2.2 Solid-phase extraction and GC-MS analysis ………………8
2.3 Isolation and characterization of ABC transporter subfamily G……8
2.4 Comparative transcriptomics and gene-to-gene correlation analysis…………9
2.5 Primer design and molecular cloning of PbABCG1 to PbABCG6…………………9
2.6 Quantitative real-time PCR (qPCR) …………………………………10
2.7 Virus-induced gene silencing and Gateway reaction preparation…………10
2.8 Bacteria injection of plants…………………………………10
2.9 PbABCG1 and PbABCG2 expression in mutant yeast ……………11
3.RESULT……………………………………………………………………………13
3.1 Identification of ABCG genes in P. bellina……………13
3.2 Phylogenetic analysis of the 6 candidate ABCGs……………………14
3.3Correlation analysis according to gene expression profiles by using ContigViews…………………………………………………14
3.4 Temporal and spatial gene expression of the candidate PbABCGs by qPCR analysis……………………………………………14
3.5 Functional characterization of PbABCGs by VIGS………………16
3.6 Functional characterization of PbABCGs using RNAi…………17
3.7 Complementation of both PbABCG1 and PbABCG2 in mutant yeast for resistance    to monoterpene toxicity……………………………18
4.DISCUSSION……………………………20
4.1 PbABCG1 and PbABCG2 belongs to the PDR transporters…20
4.2 Comparison of VIGS and RNAi inhibition effects…………………21
4.3 Differences in inhibitory effects of VIGS and RNAi on plants……22
4.4 The performance of ABC transporters on yeast…………23
5.CONCLUSION and PERSPECTIVE……………………24
REFERENCES…………………………………………25
Table……………………………………………29
Figure……………………………………………31
Supplementary Figures…………………………………………50
List of Table
Table 1. ABCG genes involved in transport or secretion of terpenoids or alkaloids………29
Table 2. Sequence of primers used in this research…………………30
List of Figure
Figure 1. Flower morphology………………………………31
Figure 2. Phylogenetic tree showing the relationship of ABC transporters identified in P.bellina transcriptomes with the ABC transporters from Arabidopsis…………32
Figure 3. Comparison of the expression levels of ABCG genes between two Phalaenopsis transcriptomes………………………33
Figure 4. Multiple alignment of the conserved domain of known PDR transporters involved in terpenoids transport…35
Figure 5. Phylogenetic analysis of the putative PbABCGs with known function ABCG genes from various plants showed that they were clustered into pleiotropic drug resistance (PDR) transporters (PbABCG1 and PbABCG2), and white-brown complex (WBC) transporters (PbABCG3-6) ……………36
Figure 6. Gene-to-gene correlation analysis according to gene expression profiles using ContigViews…………………37
Figure 7. Expression of floral scent emission, temporal gene and spatial gene in P.  bellina……………………………………38
Figure 8. Expression of PbABCB1 in P. bellina ……………40
Figure 9. Functional analysis of both PbABCG1 and PbABCG2 by using TRV-based VIGS…………………………………………………41
Figure 10. Detection of TRV2 in VIGS plants…………43
Figure 11. Functional analysis of both PbABCG1 and PbABCG2 by using RNAi………44
Figure 12. Growth inhibition effect of geraniol, a monoterpene, on ABC16- mutant yeast strains expressing PbABCG1 and PbABCG2……………………46
Figure 13. Silencing effects on linalool and geraniol in VIGS and RNAi plants…………48
Figure 14. Proposed model for linalool and geraniol transport in P. bellina……………49  
List of Supplementary Figure
Supplementary Figure S1. Proposed models for volatile organic compound (VOC)trafficking in plant cells……………50
Supplementary Figure S2.　in situ localization of PbABCG1 and PbABCG2 in the basal regions of sepal and petal on D+5…………………………51
                                    

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