參與相思樹黃酮醇生合成之酵素基因選殖與特性分析

Abstract

臺灣相思樹（Acacia confusa）之葉、花、枝條、樹皮與心材含有豐富的黃酮 類化合物，並且具有良好的抗氧化活性，這些黃酮類主要為黃酮醇化合物。本研 究中，以聚合酶鏈鎖反應及 cDNA 末端快速擴增法，由相思樹葉片獲得查爾酮合 成酶（AcCHS）、查爾酮異合酶（AcCHI）以及黃酮醇合成酶（AcFLS）基因全長，此三基因之核酸序列轉譯區長度分別為 1170 bp、720 bp 以及 996 bp，對應產生43 kDa、26 kDa 和 38 kDa 的蛋白質。以鄰近連接演化樹分析法（Neighbor-joining）分析親緣關係，發現 AcCHS、AcCHI 以及 AcFLS 分別被歸類於 CHS 超級基因家族中的 CHS、非豆科專屬的 Type I CHI 以及 2-ODD 超級家族中的 FLS。經過反轉錄聚合酶連鎖反應檢測，發現相思樹小苗 AcCHS，AcCHI 以及 AcFLS 的表現在葉組織最佳；而成齡相思樹之 AcCHS，AcCHI 以及 AcFLS 在花的表現最爲明顯。此外，爲了鑑定 AcFLS 之活性，本研究以高效液相層析儀檢測此蛋白質體外反應之產物。首先將 AcFLS 建構在 His-tag 系統之表現載體 pET-21a（+），並轉型至大腸桿菌中進行大量蛋白質表現，經過不同的基質檢測結果，AcFLS 可將二氫楊梅素、二氫檞皮素以及柚皮素分別轉化成楊梅素、檞皮素以及山奈醇。本研究為相思樹首篇有關於黃酮醇生合成途徑之報告，對未來的研究將可提供有用的參考價值。

The flavonoids are abundant in the leaves, flowers, branches, bark and heart wood of Acacia confusa, which possesses a remarkable antioxidant activities. The major class of compounds of the flavonoids is flavonols. In this study, polymerase chain reaction (PCR) and rapid amplification of cDNA end (RACE) were used to obtain full sequence of AcCHS, AcCHI, and AcFLS, which have 1170 bp, 720 bp and 996 bp coding region respectively. The molecular weights (Mw) of these three enzymes are 43 kDa, 26 kDa and 38 kDa, respectively. AcCHS belongs to the CHS group of the chalcone synthase superfamily; AcCHI belongs to the non-legume Type I CHI; AcFLS belongs to the FLS group of 2-ODD superfamily. In the reverse transcription-PCR(RT-PCR) test for the expression of AcCHS, AcCHI, and AcFLS, these three genes showed the best expression in the leave of the seedling, and in the flower of the mature tree. To characterize the corresponding enzyme of AcFLS in vitro, AcFLS is constructed into vector pET-21a (+) of histidine tag (His-tag) fusion protein system, and expressed in Escherichia coli for AcFLS recombinant protein. In the enzyme activity analysis, we found that AcFLS could transform dihydromyricetin, dihydroquercetin and naringenin into flavonol myricetin, quercetin and kaempferol, respectively. This is the first study of flavonols biosynthesis pathway in A. confusa, and the result might provide a useful reference for the future studies.