利用農桿菌媒介轉殖法改造大豆種子組成分

Abstract

儘管學者們對於大豆基因轉殖進行許多研究，而且抗殺草劑轉基因大豆Roundup®已經商品化大量生產，但由於品種間的遺傳變異及其對組織培養和轉殖過程的差異性反應，至今大豆基因轉殖仍然是一項挑戰。影響基因轉殖的因子相當多，包括農桿菌的品系、培植體的種類及環境等因素。本研究利用農桿菌媒介法轉殖大豆種皮CHI3基因之反義股，探討添加acetosyringone（AS）、抗生素篩選時期及傷害處理對芽體再生及轉殖效率之影響。在添加AS及抗生素篩選時期的試驗，雖然有助於芽體的再生，但對於轉殖效率並沒有顯著的影響；而傷害處理試驗，雖然芽體再生並無影響，但以電線進行傷害處理確實會提升轉殖效率，結合本研究的各種最佳處理條件，期望建立一穩定及高效率的大豆基因轉殖系統。轉殖後培植體經kanamycin抗生素篩選及PCR方法鑑定後，共獲得6株轉殖株，轉殖效率為0.7%。 利用轉基因植物生產重要代謝產物，或進行特定代謝物之改造與生產，做為抗體或疫苗，是目前生物技術中熱門且具高價值的領域，大豆含有大量的蛋白質，是作為生產重組蛋白極佳的材料。心臟血管疾病為近年來國人十大死因之一，高血脂症會增加心血管疾病發生的機率，因此血脂的控制應是預防心血管疾病首要的工作。活性胜肽Val- Val- Tyr- Pro (VVYP)具降血脂的作用，本研究運用農桿菌媒介法，以大豆子葉節為培植體，轉殖經改造後帶有活性胜肽VVYP之大豆儲藏性蛋白glycinin Gy5基因，及可抑制原有儲藏性蛋白表現的RNAi構築，期能獲得在不影響大豆種子正常發育的情況下，可提高外來基因及目標蛋白質之表現量的轉殖大豆品系，轉殖後培殖體經hygromycin抗生素篩選及PCR方法鑑定後，在Gy5-10vp基因構築部分，獲得5株轉殖株，轉殖效率為1.4 %。RNAi構築的部份， Gy1p-Gy2/3i基因構築獲得有6株轉殖株，其轉殖效率為1.7 %。

Many researches on soybean transformation have been reported and commercialized transgenic soybean plants have been planted worldwide yet the transformation of soybean is still a challenge due to the genetic variations of cultivars in tissue culture, regeneration, and transformation. Many factors that may affect soybean genetic transformation including agrobacterium strain, type of explants, and environmental conditions, etc. In this study the effects of acetosyringone (AS), stage of antibiotic selection, and wounding treatment on shoot regeneration and transformation frequency were studied using antisense CHI3 clone through Agrobacterium-mediated transformation method. No significant effect on shoot induction and transformation frequency was observed in either the addition of AS or stage of antibiotic selection. Only wounding treatment by electric wires increased the transformation efficiency. Six putative transgenic plants with antisense CHI3 gene were confirmed by PCR resulting 0.7% transformation frequency. Soybean is a good material for the production of secondary metabolites and recombinant proteins through genetic engineering because of its seed characteristics. The bioactive peptide Val-Val-Tyr-Pro (VVYP) was found to be effective in reducing the blood cholesterol and can be applied to prevent the cardiovascular diseases. Gene constructs contain soybean storage protein glycinin gene, Gy5, carrying 10 copies of VVYP, and RNA interference construct for blocking the of endogenous storage protein expression were transferred into cotyledonary nodes of soybean CWRD variety through Agrobacterium- mediated method in attempt to establish a system using the soybean as a bioreactor to produce bioactive peptides without interfering its seed proteins production. In this study, 5 and 6 putative transgenic plants with Gy5-10vp and Gy1-Gy2/3i gene were confirmed by PCR, and showed 1.4%. and 1.7% transformation frequency, respectively.