請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95717
標題: 青花菜蘿蔔硫素生合成相關基因之構築及轉殖
Construction and Transformation of Sulforaphane Biosynthetic Related Genes of Broccoli
作者: 林泳伸
Yung-Shen Lin
關鍵字: 青花菜
甘藍
基因轉移
蘿蔔硫素
代謝工程
Broccoli
Cabbage
Gene Transformation
Sulforaphane
Metabolic Engineering
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摘要: 十字花科蔬菜,例如青花菜、甘藍、花椰菜,含有高量的蘿蔔硫素(sulforaphane),其具有抑制癌細胞繁殖、防止癌細胞的發展、抗癌細胞轉移等作用。蘿蔔硫素是一種異硫氰酸鹽(isothiocyanate),由硫代葡萄糖苷(glucosinolates)經存在於植物體液泡內的黑芥子酶(myrosinase, MYR)水解而得。研究顯示ESP (epithiospecifier protein, 上皮硫特異蛋白)會抑制蘿蔔硫素的合成,ESM (epithiospecifier modifier, 表皮特異硫蛋白修飾子)則促進蘿蔔硫素的生成。由於蘿蔔硫素的萃取效率極低及高價格,限制其在醫藥的應用。 甘藍是世界重要經濟蔬菜,是台灣栽培面積最廣的葉菜類蔬菜,青花菜是十字花科作物中最具有抗癌效果的蔬菜,二者有其重要的民生及經濟地位。利用基因轉殖作物為生物反應器以生產工業、食品、飼料及醫藥方面的產品,在高附加價值、低成本及環保因素考量之下,是台灣極適合積極研究發展的重要項目。 研究理念是將自青花菜分離之MYR及ESM基因構築到蕓苔屬葉綠體基因轉殖載體系統,ESP基因構築到RNAi的之農桿菌介導的基因轉殖系統,將此三個基因各別或共同轉移至青花菜或甘藍之葉綠體/細胞核中,達到抑制ESP基因表現或/及葉綠體大量合成MYR及ESM,且區格MYR及ESM在葉綠體中,如此當植物器官組織受到傷害時,即可大量合成蘿蔔硫素。 本研究分別構築帶有青花菜Myr及Esm基因為目標基因之蕓苔屬葉綠體轉殖載體與帶有青花菜Esp基因為目標基因之RNAi抑制方式(ESPi)的農桿菌基因轉殖載體。總共完成六種蕓苔屬葉綠體轉殖載體:pMT91t-Esm-A、pMT91t-Myr-A、pMT91t-Esm-Myr-A、pMT91t-Esm-GA、pMT91t-Myr-GA及pMT91t-Esm-Myr-GA,以aadA為篩選基因或/且以gus為報導基因。完成二種RNAi抑制方式的農桿菌基因轉殖載體﹕p1304-Esp-IN-GH及p1304-Esp-IN-GD,以mgfp-gusA為報導基因,以hptII或daao為篩選基因。 本研究將pMT91t-Esm-Myr-GA載體,利用基因槍法將其轉移至''初秋''甘藍及''綠王''青花菜下胚軸中。再生培植體經10 ppm spectinomycin持續篩選,已獲得再生殖株。轉殖再生植株葉片之PCR、RT-PCR及qrt-RT-PCR分析之結果顯示,轉殖之Esm、Myr基因已存在於轉殖植株之葉綠體基因組,並表現其mRNA。
Cruciferous vegetables, such as broccoli, cabbage, and cauliflower, are rich sources of sulforaphane. Sulforaphane have been found to inhibit proliferation of cancer cells, prevent cancer cells to develop, and anti-metastasis of cancer cells. Sulforaphane is an isothiocyanate which is produced from glucosinolates in myrosinase-catalyzed hydrolysis. Myrosinase (MYR) is physically separated from glucosinolates in intact plant cells. Production of sulforaphane is upregulated by the MYR and ESM (epithiospecifier modifier) and downregulated by ESP (epithiospecifier protein). The complicated separation and purification procedure result in low extraction efficiency and extremely high price for the sulforaphane, which hampers a wide application in pharmaceutical science. Cabbage (Brassica oleracea L. var. capitata L.) is one of the most important vegetable crops grown worldwide, and also has been the most widely cultivated leafy vegetables in Taiwan. Broccoli (Brassica oleracea var. italica Planck) is well established as the best anti-cancer vegetable among the cruciferous vegetables. Using transgenic crops as bioreactors to produce industrial, feed and fodder additives, and pharmaceutical proteins become a new way for increasing economic values of crops and may solve the problems we are facing. By using the art of genetic engineering, we attempt to modify the glucosinolate-myrosinase substrate-enzyme system. Increasing MYR and ESM expression and knockdown the expression of ESP are achieved by chloroplast gene transformation and RNAi manipulation, respectively. Thus, overexpression of MYR and ESM in chloroplast and suppression of ESP expression, thorough breakdown of modified broccoli and cabbage will enhance the production of sulforaphane. In this study, Brassica chloroplast transformation vectors harboring the MYR and ESM genes were constructed. Vectors harboring antisense--oriented ESP gene (ESPi) were also constructed for Agrobacterium-mediated transformation. Six Brassica chloroplast transformation vectors had been constructed, namely pMT91t-Esm-A, pMT91t-Myr-A, pMT91t-Esm-Myr-A, pMT91t-Esm-GA, pMT91t-Myr-GA and pMT91t-Esm-Myr-GA. Two RNAi constructs for ESP gene has been obtained, namely, p1304-Esp-IN-GH and p1304-Esp-IN-GD. Brassica chloroplast transformation vectors, pMT91t-Esm-Myr-GA (harboring Myr and Esm gene), was transferred into the hypocotyls of ''K-Y cross'' cabbage and ''Green King'' broccoli via biolistic bombardment. Transformed plantlets are selected by 10 ppm spectinomycin. The results of PCR, RT-PCR, and qrt-RT-RCR analysis indicated that the transformed genes are present in the chloroplast genome of transplastomic plants, and expressed its mRNA.
URI: http://hdl.handle.net/11455/95717
文章公開時間: 2020-08-28
顯示於類別:園藝學系

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