植物特有的兩個蛋白質醣基轉移酶α-1,3-fucosyltransferase 和β-1,2-xysyltransferase對水稻的生理以及發育的影響以及開發偵測植物轉殖基因之生物探測器

利用植物系統生產重組蛋白擁有如低成本、高生物安全性、以及完整的後轉譯修飾系統等等諸多的優點。多種不同的重組細胞激素、疫苗、抗體以及其他的蛋白質已經成功透過植物系統被生產出來，而水稻因為其基因體已被解碼，輔以成熟的轉殖系統以及農作物的大幅產量，被視為一個非常優秀的系統。然而，由於植物特有的醣基化系統可能造成人類免疫系統產生過敏反應，所以有必要進一步對生產重組蛋白所使用的轉殖植物系統進行進一步的修改，使其醣基化系統更接近人類的醣基化系統。醣基化的修飾一個最大的隱憂就是它可能會影響轉殖植物的生長以及發育。在前人的研究裡，缺乏一個位於內質網和高爾基體之間負責蛋白質醣基化的基因N-acetylglucosaminyltransferase I (GnTI)之突變株生長發育相較於野生種的水稻皆為緩慢，而在幼苗時期的死亡率也大幅度的上升。我們培養出了兩個獨立的單一RNA干擾 (RNA interference, RNAi) 細胞系, 成功的分別的降低了細胞中α-1,3-fucosyltransferase 以及β-1,2-xylosyltransferase 的表現量. 此二細胞系被命名為 FTi 以及 XTi ，也被重生成植株和養成懸浮細胞. 不論FTi (T4) 還是 XTi (T3)的植株，在株高、穗數、孽數、種子數、以及種子重量都較野生種來的低。其中FTi更有兩個植株有表現出像是葉片提早的老化以及捲曲等明顯異於野生種的性狀。至於懸浮培養細胞，當蔗糖被利用來做為培養基的碳源時， FTi 和XTi的生長情況和野生種沒有顯著差異性。然而當澱粉被當成主要碳源時，兩個轉殖細胞系的澱粉降解率皆較野生種來的緩慢。然而在高鹽的逆境下，兩個轉植系的細胞生長速度和野生種沒有顯著的差異。本論文之第二部分對於已存在之水稻轉殖技術以及生產的重組蛋白做了一個整理，並且針對幾個能夠改進的部分進行說明以及探討。最後，本論文使用美國聖母大學張學嘉教授實驗室所開發的一個新型生物晶片進行水稻轉殖基因的探測. 希望經過改進後能在偵測基因改造生物以及疾病上有所貢獻.

關鍵字

水稻；醣基化； β-1,2-xylosyltransferase ； α-1,3-fucosyltransferase ；重組蛋白；生物探測器

並列摘要

Application of plant expression systems in the production of recombinant proteins has several advantages such as low maintenance cost, absence of human pathogens, and possession of complex post-translational glycosylation capabilities. Plants have been successfully used to produce recombinant cytokines, vaccines, antibodies, and other proteins, and rice (Oryza sativa) is a potential recombinant protein expression system used. However, plant-specific glycans, β-1,2-xylose and α-1,3-fucose are possible sources of human allergy, indicating that further modification in transgenic plant may be required to humanize recombinant therapeutics produced in plants. In order to study the effects of knockdown of β-1,2-xylosyltransferase and α-1,3-fucosyltransferase, two enzymes responsible for the addition of β-1,2-xylose and α-1,3-fucose on plant proteins, on the physiology and development of the plants, transgenic lines expressing constitutive RNA interference of the two enzymes were developed, and numerous analysis were performed on the transgenic lines and suspension cells. Transgenic plant physiology and growth are both greatly retarded. On the other hand, growth of transgenic suspension cells is not affected by RNAi of β-1,2-xylosyltransferase and α-1,3-fucosyltransferase when sucrose is used as a carbon source. When starch is used as replacement carbon source, Single knockdown RNAi cell lines still do not differ significantly from the wild type. Triple knockdown RNAi cell lines displays reduced starch degradation rate. Next, the current progress in production of recombinant pharmaceutical proteins expressed inside plant systems is discussed, production and properties of the proteins are listed, and potential improvements such as promoter selection, codon usage, glycosylation modification, genome stabilization, and large scale production, are explored. With emerging advances in transgenic technology, it is also important to screen for transgenic organisms in order to distinguish them from wild type organisms. Reverse transcription polymerase chain reaction (RT-PCR) is usually used to detect genes of interest. However, it has several disadvantages such as time consumption and location limitation. Here, a novel biochip design is proposed in which RNA binding to a probe anchored on membrane is proposed. Detection of positive binding is achieved through measuring of depletion region caused by flow of charged particles. While numerous challenges still need to be overcome, the system will be able to offer rapid sensing of transgene and potentially be used for disease diagnosis in the future.

並列關鍵字

Oryza sativa ； glycosylation ； β-1,2-xylosyltransferase ； α-1,3-fucosyltransferase ； recombinant protein ； biosensor

參考文獻

1. Cohen, S.N.; Chang, A.C.; Boyer, H.W.; Helling, R.B., Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A 1973, 70, 3240-3244.

3. He, Y.; Ning, T.; Xie, T.; Qiu, Q.; Zhang, L.; Sun, Y.; Jiang, D.; Fu, K.; Yin, F.; Zhang, W., et al., Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci U S A 2011, 108, 19078-19083.

5. Huang, C.J.; Lin, H.; Yang, X., Industrial production of recombinant therapeutics in escherichia coli and its recent advancements. J Ind Microbiol Biotechnol 2012, 39, 383-399.

6. Yoon, S.H.; Kim, S.K.; Kim, J.F., Secretory production of recombinant proteins in escherichia coli. Recent Pat Biotechnol 2010, 4, 23-29.

7. Mattanovich, D.; Branduardi, P.; Dato, L.; Gasser, B.; Sauer, M.; Porro, D., Recombinant protein production in yeasts. Methods Mol Biol 2012, 824, 329-358.

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植物特有的兩個蛋白質醣基轉移酶α-1,3-fucosyltransferase 和β-1,2-xysyltransferase對水稻的生理以及發育的影響以及開發偵測植物轉殖基因之生物探測器

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