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Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22072
標題: 利用植物生產降血脂活性胜肽 VVYP 之研究 : 以攜有 VVYP 之大豆改造蛋白基因 Gy5 之轉殖分析
Production of bioactive peptides VVYP in transgenic plants : Using soybean glycinin Gy5 gene as VVYP carrier
作者: Lai, Yen-Hsun
賴彥勳
關鍵字: transgenic soybean;轉殖水稻;transgenic rice;bioactive peptide VVYP;活性胜肽VVYP
出版社: 分子生物學研究所
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摘要: 
Bioactive peptide Val-Val-Tyr-Pro (VVYP) plays an important role in reducing blood cholesterol by inhibiting the absorption of triglycerides in intestinal tract and enhancing the activity of hepatic triglyceride lipase to remove extra triglycerides. In order to produce large amount of VVYP in transgenic plants, constructs containing 10 copies of VVYP engineered in a soybean storage protein Gy5 gene (Gy5-10VVYP) driven by various promoters, such as the rice glutelin promoter, soybean seed-specific β-conglycinin α-subunit promoter (BCAP) and CaMV35S promoter were created. These constructs were used to transform rice and soybean through particle bombardment or agrobacterium-mediated transformation methods. In addition, an artificial miRNA construct (amiRNA159-Gy5) that could potentially be used to suppress the expression of endogenous Gy5 storage protein and to increase the levels of Gy5-10VVYP recombinant proteins in transgenic soybeans was also constructed. However, transgenic soybean has not yet been obtained. Further characterization of soybean transformation system was evaluated including the regeneration ability of hypocotyl explants, sensitivity to various concentrations of phosphinothricin (PPT) and various co-culture conditions of agrobacterium with explants which were used to set up an optimized soybean transformation conditions for Chin-Ren-Woo-Dow (CRWD), a local soybean variety. For rice transformation, 28 GluGy5VP10 transgenic rice lines were obtained and their transgenes were identified by PCR and Southern blot assays. Results showed these transgenic rice plants contain either one or two copies of Gy5VP10 insertions. Western blot analysis using anti-Gy5 or anti-His tag antibodies against total seed proteins recognized four protein bands with apparent molecular weight of 60, 50, 40, and 20 kDa respectively. Further LC-MS/MS analysis confirmed that 60, 50, and 20 kDa proteins contain peptides of VVYP and Gy5 protein. These results demonstrated that the Gy5VP10 recombinant proteins were successfully expressed in the transgenic rice. Further analysis to demonstrate the release of VVYP from recombinant proteins and their function in reducing blood cholesterol in experimental animals are necessary to validate the usefulness of this approach.

活性胜肽 Val-Val-Tyr-Pro (VVYP) 為抑制腸道吸收三酸甘油酯之重要因子，其可經由促進肝臟 HTGL (hepatic triglyceride lipase) 之活性來降低三酸甘油酯之濃度達到降血脂之作用。本研究透過基因工程改造大豆儲藏性蛋白基因 Gy5，使其攜帶有 10 套降血脂活性胜肽 VVYP 之胺基酸序列，透過農桿菌轉殖技術，將含有 VVYP 之 Gy5 基因分別轉殖大豆及稻米，使其能大量表現帶有 VVYP 之 Gy5 基因，希望透過食用含 VVYP 之稻米及大豆，經由消化道酵素分解、吸收後，達到降血脂之功效，可大幅提高稻米及大豆之附加價值。轉殖水稻部分，利用水稻種子專一性 Glutelin 啟動子；轉殖大豆部分，利用大豆種子專一性 β-conglycinin α-subunit 啟動子 (BCAP) 及持續性表現 CaMV35S 啟動子，驅動降血脂活性胜肽 VVYP 10 套之胺基酸序列。另外透過人工合成 miRNA159-Gy5 (artificial microRNA)，針對大豆內生性 Gy5 基因進行靜默，希望能抑制其原有之儲藏性蛋白 Gy5 之表現，使改造過之外來基因 Gy5-10VVYP 之表現量增加。由於大豆轉殖的困難，本研究也擬建立一個較完善、適用於本土大豆品種青仁烏豆 (Chin-Ren-Woo-Dow, CRWD) 之轉殖平台，因此針對本研究室建立之轉殖系統進行下胚軸培殖體再生測試、殺草劑 Phosphinothricin (PTT) 濃度篩選及農桿菌轉殖條件測試，並嘗試利用基因槍 (particle bombardment) 系統進行轉殖，希望能建立較完善之大豆轉殖系統。但目前仍未獲得任何大豆轉殖株。轉殖水稻部分，目前已獲得之 28 株 GluGy5VP10 水稻再生植株，經 PCR 確認，皆為轉殖株。經南方墨點法分析轉殖基因套數，大部份水稻轉殖株含有 1 或 2 套插入基因。以西方墨點法分析種子蛋白表現，在接近 60 kDa、50 kDa 、40 kDa、20 kDa 位置之蛋白，皆可被 Gy5、Anti-6X His tag 抗體所辨識，進一步利用液相層析-串聯質譜儀 (LC-MS/MS) 進行蛋白身分鑑定，在接近 60 kDa、50 kDa、20 kDa 位置之蛋白，可比對到Gy5-10VVYP 或 大豆 Glycinin 5 (Gy5) 蛋白。由以上結果確認 GlyGy5VP10 轉殖水稻種子可表現 Gy5-10VVYP。最後進一步針對水稻外源蛋白 Gy5-10VVYP 進行大量萃取純化，以利後續試驗確認 Gy5-10VVYP 可釋放出 VVYP 胜肽片段，並以動物試驗檢測其是否具有降血脂之功效。
URI: http://hdl.handle.net/11455/22072
其他識別: U0005-0802201116573400
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