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  • 學位論文

香椿單萜類合成酶基因之選殖及其功能分析

Cloning and Functional Analysis of A Monoterpene Synthase Gene in Toona sinensis

指導教授 : 黃鵬林
共同指導教授 : 杜宜殷 趙淑妙(Shu-Miaw Chaw)
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摘要


摘要 本論文選殖香椿之萜類合成酶基因,並分析其酵素功能。萜類 (terpenoids) 化合物是植物相當重要的二次代謝產物,雖然它與植物的生長發育並沒有直接的關係,但對植物與環境之間交互作用 (例如:抗寒、缺水等逆境),及植物本身防禦病蟲害是很重要的。萜類合成酶 (terpene synthase, TPS) 是萜類生合成反應過程中最關鍵的酵素。本論文描述從香椿 (Toona sinene) 葉片之中選殖出命名為TsTPS1的TPS基因,其全長為1788個鹼基對,會轉譯出595個胺基酸,預測分子量為68.82 kDa;另構築一條基因,將TsTPS1基因前端運輸蛋白 (transit peptide) 之序列切除,命名為TsTPS1-t,其全長為1617鹼基對,會轉譯出538個胺基酸,預測分子量為62.99 kDa。利用大腸桿菌分別表現前述兩條基因,將所得到的蛋白質經過酵素純化及氣相層析質譜儀 (GC-MS) 定性後,證明從香椿中所選殖出的TsTPS1-t基因會轉譯成單萜類的正檸檬精油合成酶 ( (+)-limonene synthase);TsTPS1基因表現出的酵素量低,經活性測試亦尚未測得酵素活性。以反轉錄聚合酶連鎖反應 (RT-PCR) 的試驗結果顯示TsTPS1基因在香椿根部的表現量較高。TsTPS1基因組DNA (genomic DNA) 全長2317個鹼基對,其中包含了六個顯子 (exon) 與五個隱子 (intron)。從組織的石蠟切片,觀察到香椿葉柄上有高密度的腺細胞 (約10 granules/mm2),在葉身上則未發現任何腺細胞的存在;不過我們觀察到葉片的海綿組織靠近氣孔周圍有儲存油質的細胞。分析香椿葉身正己烷萃取物顯示,香椿的幼葉片中共含31種化合物,其中2種為單萜類、22種為倍半萜類、1種為雙萜類及6種非萜類成分。

關鍵字

香椿 單萜類 萜類合成酶

並列摘要


We cloned and functionally characterized a terpene synthase gene from Chinese Mahogany (Toona sinensis). Terpenoids are one of the important secondary metabolites in plants. Although terpenoids are not directly related to the growth and development of plants, they are important for plants to defense pathogens and to interact with their environments. Terpene synthase (TPS) is the key enzyme in the biosynthesis of terpenoid compounds. A monoterpene gene, TsTPS1, was cloned from T. sinensis. The gene is 1788 bp long and encodes a polypeptide of 595 amino acids which has a predicted molecular weight of 68.82 kDa. A truncated gene, TsTPS1-t, was obtained from TsTPS1. It is 1617 bp long and encodes a polypeptide of 538 amino acids which has a predicted molecular weight of 62.99 kDa. E. coli was used to express TsTPS1 and TsTPS1-t genes. Purified recombinant enzymes were characterized by Gas Chromatograph / Mass Spectrometer (GC/MS). Our assay demonstrated that the TsTPS1-t could be translated into (+)-limonene synthase. RT-PCR test indicated that TsTPS1 is expressed more abundantly in root than other tissues. The gemomic DNA sequence of TsTPS1 is 2317 bp long including six exons and five introns. In paraffin section, we found high density of glandular cells (10 granules/ mm2) in the petiole but not in the leaf blades of T. sinensis. Many oil containing cells near the stoma were found in the leaves of T. sinensis. By extracting with hexane, the total extractants of Chinese Mahogany’s leaves contain 31 kinds of compounds, which are consisted of 2 types of monoterpenoids, 22 types of sesquiterpenoids, 1 type of diterpenoids, and 6 types of non-terpenoids.

並列關鍵字

Toona sinensis terpenoid terpene synthase

參考文獻


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