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標題: | 發展臺灣香檬香氣特徵指紋及相關基因表現量初探 Developing Aroma Profile of Fresh Leaves and Preliminary Study of Related Gene Expression of Formosa lemon (Citrus depressa Hayata) |
作者: | Yung-Yu Liao 廖永瑀 |
指導教授: | 林書妍(Shu-Yen Lin) |
關鍵字: | シークヮーサー(日),扁實檸檬,固相微萃取,季節變化,化學分類法,種內多樣性,群集分析, Shiikuwasa,Hirami lemon,Solid-phase microextraction,Seasonal varietion,Chemotaxonomy,Intraspecies diversity,Cluster analysis, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 臺灣香檬(Citrus depressa Hayata)為原生於臺灣及日本沖繩之酸味小果柑橘,果實大小與外觀與常見的小果柑橘四季橘與酸橘相似易混淆。除種間辨識問題外,臺灣香檬其種內具有多樣性,由葉片形態特徵即顯見差異,但臺灣種原間的多樣性相關研究甚少。本研究利用葉片揮發性物質特徵,建構臺灣香檬與相似種間及種內的功能性輔助分類系統。試驗選用農業試驗分所嘉義分所之臺灣香檬蒐集種原,接穗來源包括嘉試所保留品系、花蓮、奧萬大,以及日本沖繩及一日本無籽品種,其他未知來源者作為測試組,以四季橘和酸橘建構之葉片氣味特徵指標。本試驗利用固相微萃取法萃取新鮮葉片揮發性物質,以氣相層析質譜儀分析,計算各化合物組成比例作為統計分析數值。 以葉片揮發性物質組成最高比例的二者作為氣味化學型特徵,臺灣香檬是Linalool/γ-Terpinene,四季橘為Linalool/β-Pinene,酸橘則是Linalool/Thymol methyl ether,三者利用集群分析結果亦可明確區分,其中臺灣香檬與酸橘的相似性較高。探討臺灣香檬種內多樣性,種內分群則可以Linalool/γ-terpinene比例、α-Terpineol的組成比例、p-Cymene 的組成比例、Thymol methyl ether的有無作為主要區分條件,經由以上條件可分為嘉試所品系、日本族群、臺灣採集族群(花蓮、奧萬大)三群。為確認季節對葉片氣味特徵之影響,於3、7、9、12月各取樣進行分析,結果顯示季節雖影響部分葉片揮發性物質之相對組成比例,但並不影響所建立用於分群辨別的香氣特徵指標。 根據次世代定序結果並參考前人研究,挑選出與β-Ocimene synthase、β-farnesene synthase、Limonene synthase、γ-Terpinene synthase、Linalool synthase、α-Terpineol synthase相關酵素,製作引子並進行qPCR,以基因相對表現量群集分析,所繪製之集群樹與以葉片氣味揮發性物質的集群樹種間分群大致相似,但無法良好對種內區分。本研究提出以葉片氣味特徵作為分類鑑別參考依據,提供氣味應用特徵資料庫,了解臺灣香檬種原特色是臺灣香檬產業發展的重要基礎。 Citrus depressa Hayata is an indigenous citrus of Taiwan and Okinawa in Japan. The fruits of Citrus microcarpa and Citrus sunki, which are common in Taiwan, characteristic of small and sour and similar to C. depressa and they are difficult to distinguish. Additionally, populations in Citrus depressa showed diversities, having significant different between leaf-characters. However, limited numbers of C. depressa studies discussed about germplasm resource’s diversities of C. depressa in Taiwan. Here, this study established a functional classification index of C. depressa to profile the relationship between interspecies and intraspecies by volatile organic components (VOCs) of fresh leaves. Plants materials of C. depressa scion collected from different places including from Hualien, Aowanda in Taiwan and Okinawa in Japan, and other unknown resources as test samples, all of C. depressa conserved in Chiayi agricultural experiment station. C. microcarpa and C. sunki were used to be out-group. The experiment method of the fresh leaves aroma was headspace SPME with GC-MS analysis, calculating relative amount of each individual component, taking major and secondary proportion of leaves volatiles as chemotype, and the data of VOCs were calculated for cluster analysis. The chemotype of C. depressa, C. microcarpa and C. sunki were Linalool/γ-terpinene, Linalool/β-Pinene, and Linalool/Thymol methyl ether respectively. The cluster result of those samples can be distinguished, and also showed that C. depressa has closer relationship with C. sunki than C. microcarpa. The diversity of C. depressa can be profiled by the proportion of Linalool/γ-Terpinene, α-Terpineol, o-Cymene, and whether Thymol methyl ether was detected. All samples of C. depressa can separate in three groups through the above conditions. To confirm effect of season to fresh leaves aroma characteristic, sampling for analysis in March, July, September and December. The result showed the seasonal variety in several composition of VOCs, but the changes did not affect the aroma classification index. Further to analyze the relative gene expression of VOCs by qPCR, the primer which selected from the result of next generation sequencing and previous studies were related to β-Ocimene, β-farnesene, limonene, γ-terpinene synthase, linalool synthase, and α-terpineol synthase. Data of relative gene expression also calculated for cluster analysis. This relative gene expression cluster presents roughly similar position of interspecies with fresh leaves VOCs cluster, but it was able to distinguish between intraspecies well. In this research using VOCs of fresh leaves as the index of the chemotaxonomy for C. depressa was able to profile the relationship between inter- and intraspecies, providing a database of aroma application characteristics. Understanding the germplasm resource’s characteristics of C. depressa is an important foundation for the development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68494 |
DOI: | 10.6342/NTU202003729 |
全文授權: | 有償授權 |
顯示於系所單位: | 園藝暨景觀學系 |
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