Identification of potential genes/enzymes involved in biosynthesis pathway of polyacetylenes in Bidens pilosa L.

大花咸豐草 (Bidens pilosa L., BP) 屬於菊科植物，為四處可見的中草藥與主要青草茶成分之一，在傳統用途中宣稱可治療許多疾病。聚乙炔類化合物 (polyacetylene, PA)具有功能性不飽和共軛三鍵或雙鍵結構，是大花咸豐草中主要的次級代謝物之一，先前試驗已證實其具有抗血管增生及抗第一型糖尿病等功效。雖然此類化合物具藥理性與重要性，但它們在大花咸豐草中的生合成途徑仍然未知。實驗室先前試驗中發現茉莉酸甲酯 (methyl jasmonate, MeJA)會調控大花咸豐草葉子中聚乙炔類化合物的生合成，並且利用處理及未處理茉莉酸甲酯、高鹽及物理性傷害的葉子樣本建立此藥草之轉錄體學 (transcriptomics)數據與資料庫。本研究旨在探討與分析參與聚乙炔類化合物生合成相關的酵素基因。我們實驗方法是將四周大的大花咸豐草幼苗處理1 mM茉莉酸甲酯，並於處理後的第0天至第16天收集葉子與根的樣本，並利用高效液相層析儀 (HPLC)分析在處理茉莉酸16天中六種主要聚乙炔類化合物的變化。接著我們依先前在轉錄體學中組裝分析獲得的39,200條基因之RNA序列來設計DNA微陣列晶片(microarray chip) ，進行功能基因體學研究觀察處理及未處理茉莉酸甲酯的葉子與根，試圖找出與聚乙炔類化合物生合成相關的基因，再進一步由即時反轉錄聚合酶連鎖反應 (real time RT-PCR)確認我們所選的基因表現量變化趨勢。另一方面，為了確認所選的基因與聚乙炔類化合物生合成具有關聯性，我們建立了核糖核酸干擾 (RNA interference, RNAi) 技術系統，利用農桿菌轉染至大花咸豐草中，在特定時間點收集及分析樣品，並與負控制組的特定基因表現量與代謝物產物含量進行比較。其中已有四個基因Δ12-oleate desaturase (BPOD)、Δ12-fatty acid acetylenase (BPFAA)、microsomal oleate desaturase FAD2-5 (B26081)及lipase (B35957)的表現變化量因符合聚乙炔類化合物代謝物含量變化的趨勢已被選於進行核糖核酸干擾實驗以確認其功能，結果顯示BPOD、BPFAA 及 B35957基因實驗後的第六天，基因有產生靜默現象，聚乙炔類化合物代謝物含量同樣地也在第六天下降，而B26081則在進行核糖核酸干擾實驗後的第三及六天基因皆產生靜默反應，聚乙炔類化合物代謝物含量也在相同的時間點有明顯的減少。總結，本論文之研究建立了可用於探討藥用植物中活性成分之生合成途徑的策略。而此篇研究的結果發現茉莉酸甲酯可能會誘導大花咸豐草中與聚乙炔類化合物生合成有關的酵素基因，我們將再進行選出之特定酵素基因的生化功能鑑定，希望能幫助我們更了解聚乙炔類化合物之生合成途徑。Bidens pilosa L. (BP) from family Asteraceae is a cosmopolitan annual medicinal herb, known for its traditional use in treating various diseases. Polyacetylenes have been identified from B. pilosa plants showing anti-angiogenesis, anti-type I diabetes, and other bioactivities. The bioactive polyacetylenic compounds contain a unique carbon–carbon triple or double bond functionality, however, their biosynthesis in BP plant is still unknown. Previous study in our laboratory showed that methyl jasmonate (MeJA) treatment can affect polyacetylenes accumulation in BP leaves, and transcriptomics study of BP leaves with or without MeJA treatment have been completed. The objective of this study was to identify and analyze putative enzyme genes involved in polyacetylenes biosynthesis. We used the four weeks old BP seedlings to treat with MeJA and harvested the leaf and root tissues at different time points from 0 to 16 days, and the chemical profiling and contents of six major polyacetylenes were examined by high-performance liquid chromatography (HPLC). The comparative functional genomics study of the BP leaf and root tissues, with or without treating with MeJA was used the in-house designed DNA microarray chips based on the RNA-seq data containing 39,202 annotated genes to study the gene expression profiles , aiming to select out the “tool-box” genes involved in the PAs biosynthesis. The expression profiles of selected candidate genes were further confirmed by real time RT-PCR and RNAi gene knockdown experimental system through Agrobacterium biotransformation in BP plants, in parallel, the PA profiles in the control mock plant and specific gene knockdown plant tissues were compared. Our results showed that the expression profile of four genes encoded for Δ12-oleate desaturase (BPOD), Δ12-fatty acid acetylenase (BPFAA), microsomal oleate desaturase FAD2-5 (B26081) and lipase (B35957) have similar trend to polyacetylenes production profile, which were selected to perform RNAi experiment in BP plants for their function validation. The BPOD, BPFAA and B35957 genes were revealed significant reduction in their expression levels at day 6 in concomitant to the significantly decreased of PAs accumulation in BP leaf, while B26081 gene expression level was decreased at day 3 and day 6 corresponding to the significantly decreased of PA contents at the same days. In summary, the current study demonstrates an approach to investigate the biosynthesis pathway of bioactive compound in medicinal plant. The results indicate that MeJA can potentially induce specific enzyme genes involved in polyacetylenes production in Bidens pilosa plant. Further functional validation of the selected candidate genes would anticipate to help understanding the biosynthesis pathway of bioactive polyacetylenes compounds in the medicinal plant