利用植物生產降血脂活性胜肽VVYP之研究：以攜有7套VVYP之大豆改造蛋白基因Gy1之轉殖分析

Abstract

本研究之目的為利用轉殖植物來生產大量帶有活性胜肽VVYP之外源蛋白。所用之構築為利用改造之大豆貯藏性蛋白Gy1基因來攜帶7套VVYP，並透過不同啟動子來調控，而後再利用農桿菌轉殖方式分別轉殖入水稻及大豆之中。利用轉殖植物來生產帶有VVYP之外源蛋白，經食用後可直接藉由胃腸道酵素水解後釋放出VVYP來達到降血脂效果。就轉殖水稻而言，本研究分別利用水稻肌動蛋白(actin)、水稻胚特有基因Ose705及水稻貯藏性蛋白glutelin之啟動子來調控Gy1-VVYP構築，此三種構築分別獲得22、25及17株再生植株。利用PCR分析所有再生植株，除了Ose705構築之兩株再生植株不帶有轉殖基因外，其他皆帶有轉殖基因；以Southern blot分析此三構築之部份植株，其插入套數約為1~2個；RT-PCR分析部份植株之結果，亦皆可見目標基因被轉錄出來，然而在以Western blot分析蛋白表現方面，僅glutelin構築植株之成熟種子蛋白於約24、55及60 kDa處可被anti-Gy1及anti-6xHis tag抗體辨認。後續再進行LC-MS/MS分析，鑑定結果發現於轉殖株中之55及60 kDa之蛋白中皆含有來自大豆蛋白的Gy1蛋白，其中並攜帶有VVYP活性胜肽。以上結果皆顯示利用轉殖水稻確實可成功表現出帶有Gy1-VVYP之外源蛋白。後續分析則須再進一步以酵素trypsin及carboxypeptidase B剪切外源蛋白看是否可釋出VVYP，而後再透過動物實驗來證實存在之VVYP確實能夠降低血液中之三酸甘油脂含量，來確定其能成為具有功能性之食物。在轉殖大豆方面，則是分別利用來自大豆貯藏性蛋白之β-conglycinin的alpha subunit啟動子(BCAP)、來自水稻肌動蛋白(actin)啟動子，及來自大豆之ubiquitin啟動子(Gmubi)來調控帶有Gy1-VVYP基因。此三種構築雖然獲得許多再生植株，然而經分析確認所有植株皆不帶有轉殖基因；另一方面尚有一預期能夠抑制內生性之貯藏性Gy1蛋白之artificial microRNA (amiR159-Gy1)構築，然而此構築尚未獲得任何再生植株可進行分析。

The aim of this study is to produce a large amount of recombinant proteins containing bioactive peptide VVYP in transgenic plants. Constructs containing 7 copies of VVYP engineered in a soybean storage protein Gy1 gene (Gy1-7VVYP) driven by various promoters were created and they were used to transform rice and soybean respectively through Agrobacterium- mediated transformation method. The VVYP containing recombinant proteins produced in the transgenic plants could be directly uptake and digested through gastrointestinal enzymes to release VVYP and thus reach the goal in reducing the blood lipid. For rice transformation, Gy1-VVYP constructs driven by the promoters of the rice actin, embryo-specific Ose705 and glutelin genes were used and 22, 25, and 17 putative transgenic rice lines for each construct were obtained respectively. PCR analysis confirmed the existence of target gene in most of the rice lines, except 2 lines in the Ose705 construct. Southern blot analysis showed transgenic rice lines contain either one or two copies of Gy1-VVYP insertions. RT-PCR analysis demonstrated the successful expression of the target gene. However, Western bolt assay showed only the proteins extracted from transgenic lines with glutelin construct could be recognized. Three protein bands with apparent molecular weight of 60, 55, and 24 kDa could be recognized by anti-Gy1 and anti-His tag antibodies. Further LC-MS/MS analysis confirmed that 60 and 55 kDa proteins contain peptides of VVYP and Gy1 protein. These results demonstrated that the Gy1-VVYP recombinant proteins were successfully expressed in the transgenic rice. Further analysis to demonstrate the release of VVYP from recombinant proteins digested with trypsin and carboxypeptidase B and their function in reducing blood triglycerides in animals are necessary to confirm the existence of VVYP peptide and their application as functional foods. For soybean transformation, Gy1-VVYP constructs driven by the promoters of the rice actin, the soybean alpha subunit of beta-conglycinin (BCAP) and the soybean ubiquitin (Gmubi) were performed. Although many regenerated soybean plants were obtained, none of them were confirmed to contain the target gene. Another approach to create an artificial miRNA construct (amiRNA159-Gy1) that could potentially be used to suppress the expression of endogenous Gy1 storage protein was also performed. However, no such transgenic soybean plant was obtained.