部分發酵茶製程揮發性有機化合物生合成之轉錄體研究

Abstract

茶樹 (Camellia sinensis (L.) O. Kuntze) 是具經濟價值的飲料作物，在全世界廣為飲用。部分發酵茶是台灣的重要茶類，製造過程包括採菁、日光萎凋、室內萎凋、炒菁、揉捻與乾燥，適當的發酵賦予部分發酵茶特殊的花香與果香，而香氣更是影響茶葉品質與特色的關鍵因素。為了瞭解茶葉中與揮發性有機化合物生合成相關的酵素，及其在製造過程中的變化，於茶菁採收前及製茶過程中的六個時間點進行採樣，以次世代核醣核酸定序分析技術進行轉錄體定序。於全新序列組裝後，共得到 222,087 條鄰接序列 (contig)，與已知基因序列比對後，其中 29% 完成基因名稱、開放讀框 (open reading frame) 與基因本體註解 (Gene Ontology term)。接著去除重複性序列，得到 20,137 條已註解的特定基因 (unigene)，其中共有 1,237 個基因於製茶過程中具差異表達，18 個特定基因註解到參與揮發性萜類、脂肪酸與β-胡蘿蔔素衍生揮發性化合物之生合成相關酵素，而乙醇脫氫酶 (alcohol dehydrogenase) 為茶葉製程前期相對基因表現量提升最多的基因。由本研究結果，我們提出了茶葉製造過程中可能的揮發性萜類生合成途徑。另一方面，由於β-櫻草糖苷 (β-primeveroside) 是茶葉中糖苷鍵結之揮發性物質前驅物存在的主要形式，故搭配次世代定序結果進行β-櫻草糖苷酶 (β-primeverosidase) 基因序列之選殖與大腸桿菌異體表現，未來能進一步純化，具有以外源酵素的方式於茶葉製程增進香氣表現之潛力。綜合以上結果，本研究提供了製茶過程中，與茶葉香氣物質生合成相關的基因表現變化，及有助於提升茶葉香氣之方法。

Tea plant (Camellia sinensis (L.) O. Kuntze) is a beverage crop, with great economic value and is consumed worldwide, in which semi-fermented oolong tea is an important feature of teas in Taiwan. The particular manufacturing process of semi-fermented tea involves plucking, solar withering, indoor withering, panning, rolling and drying, gives the tea a unique floral and fruity aroma that affects the quality and character of tea. To identify enzymes related to volatile organic compounds biosynthesis and their expression profiles during early stages of tea manufacturing process, transcriptome of tea shoots from six sampling points were sequenced using next generation RNA sequencing technology. After de novo assembly, a total of 222,087 contigs were aligned with known sequences. 29% of the contigs were annotated with gene name, open reading frame, and Gene Ontology terms. After reducing redundancy, 20,137 unigenes were obtained, with 1,237 unigenes differentially expressed during tea manufacturing process. There were 18 unigenes annotated to enzymes involved in the biosynthesis of volatile terpenes, volatile fatty acid derivatives and carotenoid derivatives. Alcohol dehydrogenase was the most up-regeleted gene during early stage of tea processing, and putative volatile terpene biosynthesis pathway in C. sinenesis was proposed. On the other hand, since β-primeverosides are the major forms of glycosidically bound volatiles precursors in tea, the sequence of β-primeverosidase was cloned with primers from RNA-seq results and expressed with E. coli, which can further be purified and had a potential to use as exogenous enzyme for aroma enhancement. This study provided novel insight into tea volatiles with the expression profile of related genes during manufacturing process, as well as potential approach to improve tea flavors.