茶樹中CsSCPL基因之分子鑑定及在非生物性逆境下表現之特性探討

茶為目前在世界上最廣泛的飲品，超過三十個以上的國家皆有種植茶樹。茶樹中含有非常大量的二級代謝物，特別是多酚類及類黃酮類化合物，當中含量最多的則為兒茶素。文獻指出，喝茶時所產生的澀味主要由酯型兒茶素(gallate-type catechin)所引起。酯型兒茶素主要是由茶樹中的絲胺酸羧肽酶(serine carboxypeptidase-like acyltransferase, SCPL)反應生成。因此，本研究將從茶樹中選殖出CsSCPL基因，並進一步了解茶樹中CsSCPL基因之分子特性及在外部環境變化下表現之情形。我們從青心烏龍茶樹中選殖出一個新的CsSCPL基因。該基因之cDNA全長包含一段長度為1440鹼基對的開放閱讀框，可編碼480個胺基酸序列，預測之分子量為54.5 kDa，等電點為5.25。與柿子(persimmon) DkSCPL、釀酒葡萄(grape) VvSCPL、野草莓(woodland strawberry) FvSCPL、柳橙(sweet orange) CsSCPL17等高等植物之SCPL蛋白進行序列比對分析，結果發現CsSCPL與其他SCPL蛋白具有高度同源性。利用即時定量聚合酶連鎖反應(Quantitative real time polymerase chain reaction, qRT-PCR)方法分析，發現CsSCPL基因主要表現在茶苗的嫩葉以及成熟茶樹的一心二葉。在非生物性逆境處理下，CsSCPL基因表現受到高溫所誘導，然而在低溫、高鹽或乾旱處理下其表現量下降。利用高效液相層析法(high performance liquid chromatography, HPLC)分析發現高溫環境與酯型兒茶素之生成具有正關聯性。進一步收取在同一地點但四個不同生長季節之茶樹一心二葉材料進行分析，qRT-PCR分析結果顯示，CsSCPL基因相對其它季節而言，在夏季具有最高的表現量，且基因表現量與季節氣溫之變化亦呈現相同趨勢。CsSCPL基因上游啟動子調控序列之分析結果顯示，CsSCPL基因啟動子具有多個受光線及荷爾蒙如生長素(auxin)或乙烯(ethylene)調控之保留性序列。針對茶樹CsSCPL蛋白之確切功能分析則有待更進一步研究探討，以了解其在植物二次代謝物生合成途徑扮演之角色。Tea (Camellia sinensis L.) is grown in more than 30 countries, and is the most widely consumed beverage in the world aside from water. Tea contains many secondary metabolites, especially polyphenolic compounds and flavonoid. The main substances causing astringent taste in oolong tea are catechins. It has been shown that gallate-type catechins are far more astringent than non-gallate-type catechins. Previous research had shown that the Serine carboxypeptidase-like (SCPL) proteins have involved in galloylated catechin biosynthesis. We isolated a CsSCPL gene encoding a serine carboxypeptidase-like protein from oolong tea plant. The full-length CsSCPL cDNA contains a 1440-bp open reading frame, encoding a protein of 480 amino acids with a calculated molecular mass of 54.5 kDa and an isoelectric point of 5.25. Sequence alignment analyses showed that CsSCPL is a serine carboxypeptidase with high homology to other SCPL proteins, including DkSCPL (persimmon), VvSCPL (grape), FvSCPL (woodland strawberry), and CsSCPL17 (sweet orange). Quantitative RT-PCR analyses revealed that the highest transcript levels of CsSCPL were in young leaves of tea seedlings and in buds of mature tea plants. The CsSCPL transcript levels increased in response to heat stress but decreased in response to cold, high salinity, and drought stresses. The degree of catechin galloylation was positively correlated with CsSCPL transcript levels after heat treatments. During four growing seasons, the CsSCPL was presented highest transcript levels in summer, which has the highest mean monthly temperature in the field. In silico promoter analysis presented that the CsSCPL promoter contains several important cis-elements which regulated by light, heat, cytokinin, and ethylene. Our results may provide useful information for further research on SCPL function in plants and oolong tea manufacture in the future.中文摘要.................................................i 英文摘要............................................... ii 目錄................................................. iii 壹、前言.................................................1 一、茶葉中主要成分介紹....................................1 二、茶葉中兒茶素之功效....................................2 三、環境逆境對茶葉二次代謝物之影響..........................5 四、SCPL醯基轉移酶.......................................7 貳、材料與方法..........................................12 一、實驗材料............................................12 二、總RNA之萃取.........................................12 三、第一股cDNA之合成.....................................13 四、TBE瓊脂凝膠(agarose)膠片配製.........................14 五、載體構築............................................14 六、即時定量聚合酶鏈鎖反應分析............................18 七、茶樹CsSCPL基因之選殖.................................19 八、CsSCPL 基因生物資訊學分析............................20 九、茶苗非生物逆境處理...................................21 十、CsSCPL基因上游啟動子序列選殖..........................22 十一、CsSCPL重組蛋白之表達...............................23 參、結果................................................25 一、茶樹SCPL基因之選殖...................................25 二、茶樹SCPL cDNA序列與胺基酸序列之生物資訊學分析..........26 三、CsSCPL基因在茶苗與茶樹不同組織中之表現情形.............27 四、茶苗CsSCPL基因及酯型兒茶素在非生物逆境下之表現量........28 五、一年四季氣溫變化對茶樹CsSCPL基因表現量之影響............29 六、茶樹CsSCPL基因啟動子序列選殖及序列分析.................30 七、茶樹CsSCPL蛋白質在Escherichia coli之表現.............31 肆、討論...............................................33 一、茶樹CsSCPL基因之鑑定................................33 二、茶樹CsSCPL基因表達部位之探討.........................34 三、非生物性因子對茶樹CsSCPL基因與兒茶素生成之影響.........34 四、調控茶樹CsSCPL基因表現之探討.........................36 五、鑑定茶樹CsSCPL蛋白之功能.............................37 伍、參考文獻............................................39 陸、圖表................................................51 柒、附錄................................................6