牛樟芝之類黃酮類化合物相關基因選殖及其特性分析

Abstract

牛樟芝（Antrodia cinnamomea）為台灣特有的平伏型擔子菌，只生長於台灣保育類樹種牛樟樹的樹心空胴處。傳統中藥裡牛樟芝在民間被視為珍貴的藥材，被認為具有保肝、解毒、抗發炎及抗氧化等功效。而本研究即針對牛樟芝尚未被探討的具強抗氧化能力之類黃酮類生合成基因及產物進行篩選及功能分析。首先，遂由應用真菌研究室所建構及註解之牛樟芝cDNA基因資料庫，顯示三個基因和類黃酮類生合成相關，並由基因片段EST（expresstion sequence tag）設計引子對選殖出flavonol synthase（FLS）、flavonoid 3’ and 5’ hydroxylase（F3’5’H）及chalcone isomerase（CHI）三基因。此外，未被註解之其他類黃酮類生合成基因，如:flavanone 3-hydroxylase （F3H）及4-coumaroyl CoA ligase （4CL）等基因則設計簡併性引子對（degenerate primer）進行選殖，其後將此等基因探針呈雜合反應之fosmid clones進行primer walking，以求得genomic DNA全長，其後預測並進行reverse transcription以選殖cDNA全長。此等基因之生化特性亦經由相關之生物資訊網站加以分析。其後利用反轉錄牛樟芝之total RNA，將類黃酮類生合成路徑的5個基因cDNA全求得並建構在Kluvyromyces lactis之表現載體pKLAC1上，以利轉型至K. lactis乳酸酵母菌系統大量表現，並期望針對基因轉殖之K. lactis產物進行抗氧化及抗癌之活性測試，並希冀應用於臨床藥物與保健食品之領域。另一方面，針對A. cinnamomea菌絲可能產生之類黃酮進行甲醇粗萃取物分析，藉由高效液相層析儀（HPLC）及600 MHz核磁共振儀（NMR）檢測，結果顯示粗萃取物中以醣類居多，而環狀結構化合物則所佔比例不高，相對地推測類黃酮類在菌絲中含量不高，未來期望藉由基因層級於K. lactis大量表現類黃酮類產物並進行特性分析。

Antrodia cinnamomea （Neu-Chang-Ts） an unique endemic resupinate basidiomycete of Taiwan, inhabited on the heartwood cavity of Cinnamomum kanehirae, reputed for alleviating, hangover, inflammation, the hepatitis syndrome and oxidative stress, etc. Attempt to clarify the antioxidation activity of A. cinnamomea, we cloned five flavonoid biosynthesis genes: flavonol synthase （FLS）, flavonoid 3’ and 5’ hydroxylase （F3’5’H）, chalcone isomerase（CHI）, flavonone 3-hydorxylase （F3H） and 4-coumaryl CoA ligase （4CL） via the constructed cDNA and fosmid libraries and PCR approaches. Furthermore, we also worked out the full length of gDNA and cDNA of these genes and their properties characterized. Currently, these genes have been constructed on pKLAC1 shuttle vectors and transformed them into Kluyveromyces lactis, host were undergoing. The expressed secondary products will be assessed and assayed. In other aspects, to clarify wether the existence of flavonoid in A. cinnamomea, the methanolic extracts from mycelium were subjected to high performance liquid chromatography（HPLC）, nuclear magnetic resonance（NMR）analysis. The outcomes indicated carbohydrates, instead of flavonoid-like substance, dominated the most partially purified fraction. Given flavonoid processes major anti-inflammation and antioxidation capacity, perspectively, overexpress the genetically engineered flavonoid biosynthetic genes in pertinent hosts to produce adequate quantify of desired compounds for clinic trals will be the resolution.