楓香葉變色分子調控機制之研究

Deciphering the Molecular Regulation Mechanism of Leaf Coloration in Formosan Sweet Gum (Liquidambar formosana Hance)

10.6342/NTU201701237

文起祥

楓香 ； 葉變色 ； 葉老化 ； 基因調控 ； 花青素 ； 原花青素 ； Liquidambar formosana Hance ； leaf coloration ； leaf senescence ； gene regulation ； anthocyanin ； proanthocyanin

國立臺灣大學森林環境暨資源學系學位論文

2017年

博士

曲芳華

繁體中文

落葉性樹種在秋冬改變葉片顏色的現象長久以來為世人熟知。相較於作物及花卉，以景觀為主的秋季葉變色現象（autumn leaf coloration）研究直到近年才因為氣候變遷議題而逐漸得到重視。本研究以臺灣原生樹種楓香（Liquidambar formosana Hance）為材料，使用其秋季葉變色延續時間較長及表現型多樣等特性，透過次代定序（next-generation sequencing）資料庫建立、基因功能鑑定及主要色素成分分析，嘗試解釋落葉性喬木葉片變色的基因調控網絡。 研究材料為臺灣大學校園內之楓香行道樹，選定一株為主要樣本來源，代表常見的表現型並以「普通型」命名之。此外輔以「紅葉型」、「黃葉型」及「常綠型」三種表現型之個體，進行2011至2014連續四年生長季每月一次的取樣。轉錄體（transcriptome）資料庫由2010年12月之樣本及2011年4月之樣本分別解序後進行混合組裝，同一樣本也進行降解體（degradome）及small RNA解序。基因表現資料庫使用2011至2013年4月、6月、8月、10月及12月樣本，透過由轉錄體組裝之序列客製設計微矩陣生物晶片（microarray）進行表現量分析。以基因共表現網絡分析（gene co-expression network analysis）的結果，預測楓香秋季葉老化中重要的轉錄因子及這些基因與葉變色相關酵素的調控關係。 前人研究顯示葉片呈現紅色或紫紅色多為花青素苷（anthocyanins）累積所致，在基因共表現網絡中，選擇具跨物種保守的花青素專一性MYB基因LfMYB113進行基因功能鑑定。在LfMYB113過表現基因轉殖菸草（Nicotiana tabacum）實驗中，經real-time PCR證實菸草黃酮類生合成途徑中許多基因表現量皆顯著上升，尤其是生合成途徑下游的花青素相關基因。暫時性過表現LfMYB113的圓葉菸草（Nicotiana benthamiana）有肉眼可見之顏色改變，以啟動子序列證實LfMYB113能調控楓香之花青素生合成相關基因LfDFR1及LfDFR2。雖然在菸草轉殖植株沒有發現葉片加速老化的情形，但暫時性表現LfMYB113於圓葉菸草可促進葉老化相關基因的表現，說明LfMYB113可能促進葉片老化，LfMYB113成為各物種花青素專一MYB基因中首先被發現具有促進老化功能者。此外透過暫時性過表現實驗，LfMYB113被證實可以調控葉綠素降解基因LfSGR，是故LfMYB113也是第一個被證實參與葉綠素降解調控的MYB基因。 本研究初步鑑定楓香葉片含有兩種黃酮醇（flavonol）及兩種花青素（anthocyanidin）結構，在春季為黃酮醇較多，而冬季則是花青素為主。透過基因調控網絡分析及LfMYB113的功能鑑定，說明LfMYB113為楓香秋季葉老化及葉變色的調控因子，其基因表現可能是造成楓香不同表現型的主要原因之一。受LfMYB113調控的LfDFR1及LfDFR2有不同的酵素活性，可能影響花青素的種類。除此之外，以LfDFR1基因表現趨勢預測，選殖可能調控花青素的候選MYB基因LfMYB123，並經功能鑑定確定LfMYB123可促進LfDFR1於春季表現。說明春季及秋冬分別由LfMYB123及LfMYB113調控花青素生合成。 在降解體分析過程中，參考其他物種研究篩選出lfo-miR828及可能受其調控的轉錄因子LfMYB5及LfTT2。經暫時性表現實驗，確定表現lfo-miR828 precursor能抑制LfMYB5及LfTT2表現，此外lfo-miR828也能造成LfMYB113及LfMYB123表現減弱。暫時性表現實驗顯示LfMYB5及LfTT8可調控原花青素生合成酵素LfLAR，而LfTT2則可能調控LfDFR2。配合LfMYB123及LfTT8調控原花青素生合成酵素LfANR，說明楓香葉變色可能受到lfo-miR828調控，以致於在lfo-miR828表現較強的夏季沒有葉變色的情形。在跨物種保守的microRNA基因分析結果中，發現楓香具有葉老化調控基因lfo-miR164，經降解體預測可調控LfNAC1，可能在楓香秋季葉老化中扮演重要的調控角色。 總結以上，透過基因之間的調控關係預測及鑑定，本研究清楚描繪楓香春季及秋季葉變色過程的主要參與基因，這些不同層級的基因包含microRNA、轉錄因子及生合成酵素，其表現結果影響黃酮類成分的種類及含量，進而影響葉片顏色的改變。

Autumnal leave coloration is one of the attractive natural events and research interests. Leaf coloration also occur when leaf sprout in the beginning of growing season, but the difference in gene regulation and pigment content remain unclear. In this study next-generation sequencing and gene function identification were used to decipher the gene regulatory network of leaf coloration in a subtropical specie Formosan sweet gum (Liquidambar formosana Hance). Formosan sweet gum leaf materials were collected on the campus of National Taiwan University. One Formosan sweet gum individual was chosen to represent the phenotype which common in Taipei, and named as “Common”. The other three phenotypes were “Red”, “Yellow” and “Evergreen”, represented different coloration phenotypes. Leaf samples were collected once a month in the growing seasons from 2011 to 2014. Samples were collected in December, 2010 and April, 2011 were used in next-generation transcriptome, small RNA and degradome sequencing. De novo assembly of reads included these two samples and produced 58,402 contigs. These contigs were used to design customized microarray. Gene expression database were constructed from microarray data with samples collected in March, Jun, August, October and December from 2011 to 2013. A putative gene regulatory network was constructed by gene co-expression analysis and revealed the link between autumn leaf senescence and coloration. Most of the autumn colors were due to the presence of anthocyanins. A conserved anthocyanin regulatory transcription factor LfMYB113 was chosen to be identified. Overexpression of LfMYB113 in tobacco (Nicotiana tabacum) confirmed the role of LfMYB113 in up-regulation of anthocyanin biosynthetic genes. Transient over-expression of LfMYB113 in Nicotiana benthamiana led visible anthocyanin accumulation in the infiltration site and it was confirmed that LfMYB113 can induced the expression of LfDFR1 and LfDFR2 during promoter assay. Although in transgenic tobacco no leaf showed pre-senescence or accelerated senescence, transient over-expression did up-regulate the senescence associated genes, indicate LfMYB113 has a role in regulation leaf senescence. In addition, it was shown that LfMYB113 induce the expression of LfSGR, and suggested LfMYB113 participated in regulation of chlorophyll degradation. In this study, it was identified that Formosan sweet gum colored leaves has two major kinds of anthocyanidins and flavonols. Formosan sweet gum leaves have more flavonols contents than anthocyanins in spring, and turns to accumulate more anthocyanins in winter. The expression of LfMYB113 may contribute to be the cause of the autumn colors. The anthocyanin biosynthetic gene LfDFR1 and LfDFR2 were shown to have different abilities in substrate reception by in vitro crude protein assay. In addition, it was found that LfMYB123 regulates LfDFR1 in spring, thus the regulation of LfDFRs by LfMYB123 and LfMYB113 may contribute Formosan sweet gum core leaf coloration regulatory mechanism. In analysis of degradome profile, it was found lfo-miR828 may repress proanthocyanin biosynthetic regulator LfMYB5 and LfTT2 in summer. The regulatory role of Lfo-miR828 was explored by transient expression experiment. The results confirmed lfo-miR828 can repress the expression of LfMYB5 and LfTT2, and partially repress LfMYB113 and LfMYB123. This suggests lfo-miR828 is a regulator in Formosan sweet gum leaf coloration. Besides, the conserve leaf senescence regulatory microRNA miR164 was also identified, lfo-miR164 may regulate LfNAC1 and thus control the autumn leaf senescence process. In summary, through the prediction and identification of the regulatory relationship of genes, this study profiled the regulatory mechanism of Formosan sweet gum leaf coloration. The genes which participated in leaf coloration included microRNA genes, transcription factors and biosynthetic enzymes. Their expression influences the content of flavonoid compounds in Formosan sweet gum leaves, and thus influence the color appearance of leaf.