臺灣杉雙萜合成酶基因之選殖與功能定性

Abstract

臺灣杉為臺灣原生的重要經濟樹種，為了解常具生物活性的雙萜類化合物於臺灣杉內之生合成機制，本研究依據基因庫中木本植物之雙萜合成酶序列設計引子對，藉由聚合酶鏈鎖反應(polymerase chain reaction, PCR)及cDNA末端快速擴增法(rapid amplification of cDNA end, RACE)獲得2553 bp的臺灣杉雙萜合成酶之轉譯區序列，以及124 bp 5’端非轉譯區、191 bp 3’端非轉譯區，並將此基因命名為 TcDiTPS；另進行基因體徒行法(genome walking)得到TcDiTPS之啟動子序列789 bp。 另一方面，本研究以reverse transcription PCR與real-time PCR觀察TcDiTPS在臺灣杉各部位組織的RNA表現狀況，結果顯示TcDiTPS在老葉與樹皮的表現量最高；同樣的方法，亦用於觀察植物處理的部分，本研究取用2-5年生的臺灣杉，剪取其老、幼枝葉，以及22天齡的濾紙苗分別進行jasmonic acid與salicylic acid的藥劑處理，結果顯示臺灣杉幼枝葉與濾紙苗中的TcDiTPS對於salicylic acid的反應大於 jasmonic acid。而TcDiTPS在老、幼枝葉5小時內的創傷處理，皆可見到對創傷有反應，其中幼枝葉對於創傷的反應速度較老枝葉快。 本研究以hisitidine tag (His-tag) 和glutathione-s-transferase (GST)融合蛋白質系統在大腸桿菌表TcDiTPS蛋白質，但從His-tag系統僅能得到不溶於水的TcDiTPS，於GST系統可得水溶性的TcDiTPS，然而與geranylgeranyl pyrophosphate反應後並無產物，推測原因為TcDiTPS在序列上缺乏完整的DXDD motif。故乃進行阿拉伯芥及臺灣杉毛狀根的轉殖，於阿拉伯芥、臺灣杉毛狀根轉殖株與野生型的代謝物差異中，推測TcDiTPS 的產物為pimara-8(14),15-diene。

Taiwania cryptomerioides (Taiwania) is an economic and endemic conifer in Taiwan. To elucidate the biosynthesis of diterpeneoids which often have bioactivity in Taiwania cryptomerioides (Taiwania), a pair of primers was designed according to the diterpene synthases of woody plant, which were collected from database. Polymerase chain reaction (PCR), rapid amplification of cDNA end (RACE) and genome walking were used for getting full sequence of Taiwania diterpene synthase. The gene was named TcDiTPS. It has 2553 bp of coding region, 124 bp of 5’ untranslated region, 191 bp of 3’ untranslated region, and 789 bp of promoter. To investigate expression level of TcDiTPS between tissues and its probably functions, reverse transcription PCR and real-time PCR were used for detection. The results showed obviously that TcDiTPS had highly expression at old leaves and bark. In treatment experiment, TcDiTPS was response to salicylic acid (SA) in both 22 days old Taiwania seedling and 2-5 years old young branches. In treatment of wounding, the expression level of TcDiTPS was increased at both young and old branches, and the response of young branches was faster than old branches. TcDiTPS was expressed in E. coli protein expression system for pure protein. It was insoluble in hisitidine tag expression system and soluble in glutathione S-transferase expression system. However, both of them could not get the diterpenoid compound after reacting with geranylgeranyl pyrophosphate. It was presumed that loss of complete DXDD motif in TcDiTPS sequence might be the key point. Another way to find the compound of TcDiTPS was using transgenic plant. According to the different metabolites between transgenic and wild-type plant, there were found diterpenoid of pimarane type and its oxidized type in the Arabidopsis thaliana and hairy root of Taiwania transgenic line, respectively. It was speculated that compound of TcDiTPS is pimara-8(14),15-diene.