Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66242
標題: Expressed Sequence Tags, Gene Cloning and Functional Characterization of Terpene Synthase in Chamaecyparis formosensis Matsum.
紅檜表達序列標籤及其萜類生合成基因的選殖與功能表現
作者: 郭佩旻
Kuo, Pei-Min
關鍵字: 表達序列標籤
Chamaecyparis formosensis
萜類生合成酶α-pinene
β-cadinene
揮發成分
茉莉酸甲酯
expressed sequence tags
terpene synthase
α-pinene
β-cadinene
volatile compound
methyl jasmonate
出版社: 森林學系所
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摘要: Chamaecyparis formosensis Matusm. is an endemic precious conifer grown in Taiwan. We used subtractive hybridization method to establish the expressed sequence tags (ESTs) in seedling and phloem of C. formosensis. Totally 384 and 452 ESTs were generated from seeding and phloem, individually. A blastx analyzed revealed that ESTs of seedling related to protein synthesis represented about 26%. The ESTs of phloem related to function of secondary metabolism and cell cycle and cell division represented about 18%. Morwover, to investigate the terpene synthesis mechanism in C. formosensis, full length of putative mono-TPS (Cf-Pin) and sesqui-TPS (Cf-Cad) were obtained by PCR method and RACE extension; and then we used recombinant protein from Escherichia coli were incubated individual with geranyl diphophate (GPP) and farnesyl diphosphate (FPP), which produced one major product, the structure of which was elucidated by gas chromatography/mass spectrometry (GC/MS) analysis. GC/MS analysis, Kovats index and MS matching with authentic standards revealed that the major products were α-pinene and β-cadinene. We cloned a mono-TPS (Cf-mono, full length: 1845 bp) and a di-TPS (Cf-di, full length: 1602 bp). We also cloned upstream of the terpene synthase including geranylgeranyl diphosphate synthase (full length of Cf-GGPPS has 1170 bp) and HMG-CoA reductase (partial sequence of Cf-HMGR has 1290 bp) of C. formosensis. To study analysis the volatile compounds of C. formosensis, in addition to the essential oil of leaves was prepared by hydrodistillation, the solid phase micro extraction technology was also conducted to collect the compounds emitted from leaves. Moreover, the leaves of C. formosensis were treated by methyl jasmonate (MeJA) to mimic it wounded or attacked by fungus or insects. The results in our study, totally 108 compounds were identified from C. formosensis, the amount of α-pinene is 30.29% is the most dominant compound in the volatile compounds emitted from leaves; however, β-myrcene (22.45%) is the most dominant compound in the essential oil. After MeJA treatment, the amount of volatile compound was increased to 1.92-fold. Meanwhile, we also found that the amount of volatiles would reach to the highest concentration after 5 hr treatment at the dosage of 10 mM MeJA.
紅檜(Chamaecyparis formosensis Matsum.)為台灣特有樹種,本研究以大量片段的DNA序列分析策略,使用扣除雜合反應建構紅檜小苗以及成熟木材韌皮部表現差異的cDNA資料庫,結果分別獲得384及452筆基因表達序列標籤,藉此探討可能之蛋白質功能及其傳達之遺傳訊息。利用blantx分析結果發現,所獲得之小苗基因功能多與蛋白質合成有關,基因數目約佔全體的26%;韌皮部之基因功能則以二次代謝作用與細胞週期和細胞分裂相關者佔較多數,約佔總數的18%。另一方面,並針對紅檜的萜類合成酶(terpene synthase, TPS)進行研究,共獲得單萜合成酶(Cf-Pin, GenBank accession No. EU099434)與倍半萜合成酶(Cf-Cad, Genbank accession No. JN715077)兩個萜類合成酶,而為了進一步研究這些紅檜TPS的功能,將TPS以載體轉入大腸桿菌中大量表現重組蛋白質,經純化後Cf-Pin與Cf-Cad的重組蛋白質則分別利用前驅物GPP與FPP進行萜類合成酶的活性鑑定,所得之產物續利用氣相層析質譜分析確定其結構,經質譜分析結果及比對KI值確定重組蛋白質反應的產物為α-pinene及β-cadinene。此外,我們亦獲得一條推測為單萜合成酶(Cf-mono,基因全長為1845 bp)與一條雙萜合成酶(Cf-di,1602 bp)。同時針對萜類合成的相關合成酶也選殖到上游的geranylgeranyl diphosphate synthase (Cf-GGPPS全長基因1170 bp)與HMG-CoA reductase (Cf-HMGR片段基因1290 bp)。在揮發性成分的研究方面,本研究除利用水蒸餾法製備精油外,並利用固相微萃取技術收集葉子的揮發性成分,續以氣相層析質譜鑑定成分。此外,利用茉莉酸甲酯模擬葉子受到機械創傷或是昆蟲、真菌的侵害後,其揮發性成分的變化。結果共鑑定出108種化合物,其中以α-pinene為最主要成分,佔30.29%;而在精油中則是以β-myrcene為主成分,佔22.45%。紅檜葉子在茉莉酸甲酯處理1 hr後,其揮發性成分之釋出量大幅提高,揮發量為未以茉莉酸甲酯處理葉子的1.92倍;而在茉莉酸甲酯處理後之經時變化,10 mM茉莉酸甲酯處理5 hr後,其揮發量增加最多,之後揮發量就開始減少。
URI: http://hdl.handle.net/11455/66242
其他識別: U0005-2208201217314900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208201217314900
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