Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22004
標題: 利用轉殖植物生產VVYP活性胜肽及特殊胺基酸組成RMGK之研究
Production of bioactive peptides VVYP and special amino acid component RMGK in transgenic plants
作者: 林家夙
Lin, Jia-Sue
關鍵字: transgenic soybean
轉殖水稻
transgenic rice
bioactive peptide(VVYP)
fish meal
活性胜肽(VVYP)
魚粉
出版社: 分子生物學研究所
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摘要: 本研究利用基因工程技術,以大豆為生物反應器大量表現活性胜肽VVYP及特殊胺基酸RMGK。活性胜肽VVYP (Val-Val-Tyr-Pro)具有降低三酸甘油脂的能力進而抑制脂肪的吸收,能有效抑制血液中三酸甘油脂的含量,減少心血管疾病的發生。在許多的雜食性淡水魚類養殖生產中,飼料配方主要來自大豆的原料已有多年的歷史,利用大豆粉取代較為昂貴的魚粉,可大幅降低魚產品的生產成本。然而與魚粉成份相比較,methionine與lysine是大豆產品中含量較缺乏的胺基酸,因此可利用基因工程在大豆基因組中加入這些胺基酸,並添加glycine及arginine增加魚類對大豆粉的嗜口性及提高魚類對疾病的抵抗性。已知固殺草 (phosphinothricin, PTT)可抑制植物體行光合及光呼吸作用而導致植物體死亡。因此本研究將選用抗PTT之bar作為篩選基因,建立大豆轉殖phosphinothricin篩選系統,以利大豆轉殖株之篩選,經由統計分析,6 及5 mg/L PTT分別為再生芽體及轉殖株後代最適當之篩選濃度。目前已經藉由農桿菌轉殖獲得135株大豆再生植株,然而以PCR及南方點墨法確認僅有一株再生植株含有轉殖基因Gy5-10RMGK,且其轉殖基因無法順利遺傳到下一代。到目前為止,由於大豆轉殖技術尚無法突破,無法得知含有VVYP及RMGK之構築是否能於植物體正常表現外源性蛋白,因此先將這些構築轉殖至水稻,進一步分析外源蛋白的表現情形,以確認此策略之可行性。利用持續性表現之35S 啟動子,構築含活性胜肽之Gy5-10VVYP及含特殊胺基酸之Gy1-10RMGK載體,藉由農桿菌進行水稻轉殖,本研究室總共獲得16株35S-Gy5-10VVYP及20株35S-Gy1-10RMGK水稻轉殖株,以PCR及南方點墨法確認這些轉殖水稻皆含轉殖基因,並以西方點墨法分析葉片及種子蛋白,確認這些轉殖水稻皆可表現重組蛋白Gy5-10VVYP或Gy1-10RMGK。
The aims of this study are to enhance the expression of bioactive peptide VVYP and special amino acid sequences RMGK in soybean and rice by genetic modification. The bioactive peptides Val-Val-Tyr-Pro (VVYP) is one of the well known effective materials to suppress the triglyceride absorption of intestinal tract and to reduce the blood cholesterol. It has been of continuing interest in developing new protein source as alternative fish meal in aquafeeds. The methionine and lysine components of soybean meal are less than fish meal. This study intends to improve the nutritional qualities by incorporating more methionine and lysine codons into a soybean storage protein gene named gy5. In addition, glycine and arginine are also added to increase the palatability of soybean meal. In this study, a transformation system using phosphinothricin (PPT) as selection marker was established and more than 135 regenerated soybean plants through agrobacterium-mediated transformation with various constructs were obtained. PCR and Southern blot assays demonstrated that only one out of 135 lines contains transgene but this transgene did not transfer to the next generation. The reason why so little transgenic line obtained and the only one identified transgenic line did not pass its transgene to the next generation is discussed in detail in the text. Since no stable transgenic soybean was obtained, the expression of engineered VVYP or RMGK-containing protein from the constructs created in this study can not be confirmed. We therefore transformed Gy5-10VVYP and Gy1-10RMGK constructs driven by CaMV35S promoter into rice to see whether or not these modified soybean storage proteins can be expressed. Sixteen transgenic lines with 35S-Gy5-10VVYP construct and 20 transgenic lines with 35S-Gy1-10RMGK construct were obtained and their transgenes were identified by PCR and Southern blot assays. Western blot analyses demonstrated that all transgenic lines tested contain modified soybean storage proteins.
URI: http://hdl.handle.net/11455/22004
其他識別: U0005-1008200922345700
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1008200922345700
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