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Title: | 以番茄與菸草為材料轉殖N-acylglucosamine 2-epimerase與N-acetylneuraminate lyase基因以生產N-acetylneuraminic acid Transforming N-acylglucosamine 2-epimerase and N-acetylneuraminate lyase genes to produce N-acetylneuraminic acid in tomato and tobacco |
Authors: | Cherng-Hann Lin 林承翰 |
Advisor: | 鄭石通 |
Keyword: | 唾液酸,番茄,N-乙醯葡萄醣胺,色胺酸合成酶, Sialic acid,tomato,N-acetylneuraminic acid (Neu5Ac),Tryptophan synthase β1 (AtTSB1), |
Publication Year : | 2011 |
Degree: | 碩士 |
Abstract: | 唾液酸是含有9個碳原子的酸性胺基醣類,在自然界有43種衍生物,氮原子乙醯化的唾液酸便是N-乙醯葡萄醣胺(N-acetylneuraminic acid)。N-乙醯葡萄醣胺在自然界分布最廣,並且是人體中最主要的唾液酸。分子結構上N-乙醯葡萄醣胺存在於糖蛋白(glycoprotein)和糖脂(glycolipid)上多醣的末端,是辨識細胞的重要標記;並且在人體中有65%唾液酸存在於中樞神經系統中。病源菌和病毒尌是以附著在N-乙醯葡萄醣胺的方式,而侵入人體細胞,因此病源菌辨識N-乙醯葡萄醣胺,並且附著寄主細胞,為寄主感病的第一個步驟。唾液酸可應用在降低病原菌和病毒辨識寄主細胞。唾液酸也是組成神經細胞的重要成分之一。但是N-乙醯葡萄醣胺不存在於植物體中,故本實驗以reverse transcription-polymerase chain reaction的方式由豬腎細胞中的cDNA中分離出可以合成N-乙醯葡萄醣胺的兩個基因,分別可產生酵素N-acylglucosamine 2-epimerase (AGE)和N-acetylneuraminate lyase (NAL),並同時將AGE和NAL建構在植物轉殖載體上,以農桿菌將此載體轉染至菸草中,並以載體上原有的抗hygromycin基因做為篩選標誌。目前已由HPLC分析得知,在轉殖菸草中,可以合成N-乙醯葡萄醣胺。同時也在含有AGE和NAL的植物轉殖載體上,加上阿拉伯芥的tryptophan synthase beta 1 (AtTSB1)基因作為非抗生素抗性篩選標的基因,以農桿菌將此含有AGE、NAL、和AtTSB1的載體轉染至番茄中,再以5-methyl tryptophan篩選得到轉殖番茄。目前致力於轉殖番茄以得到含有N-乙醯葡萄醣胺的機能性番茄。 The sialic acids are a family of nine carbon α-keto acids that play a wide variety of biological roles in nature. In mammals, they are found at the distal ends of cell surface glycoconjugates, and thus are major determinants of cellular recognition and adhesion events. N-acetylneuraminic acid (Neu5Ac) is the most widespread form of sialic acid and nearly the only form found in humans. Influenza viruses recognize the Neu5Ac on the cell surface, and then bind to host cells. The application of Neu5Ac was shown to decrease the binding of pathogens to host cells. Neu5Ac is also a key component of neural tissues. However, Neu5Ac has not been found in plants. Two genes, encoding N-acylglucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL), involved in synthesis of Neu5Ac were isolated from the cDNA of porcine kidney by reverse transcription-polymerase chain reaction. Vector containing both AGE and NAL genes were then constructed, and transformed into tobaccos by Agrobacterium-mediated transformation. The selection marker gene of the vector is the hygromycin-resistance gene, hptII. By HPLC analysis, Neu5Ac can be synthesized by the transgenic tobacco. Also, a non-antibiotic selection marker gene, AtTSB1, from Arabidopsis was introduced into the vector with AGE and NAL genes. The vector with AGE, NAL, and AtTSB1 was transformed into tomatoes by Agrobacterium-mediated transformation, and the transgenic tomatoes were selected by 5-methyl tryptophan. Now, the nutritional enhancement tomatoes with Neu5Ac is on progress to be produced. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23615 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 植物科學研究所 |
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