研究生(英)Chang, Kai-chao
論文名稱(英)Employing systems biology and RNA interference methods to dissect the physiological function of L-form starch phosphorylase in potato
指導教授(英)Chen, Han-min
關鍵字(中)澱粉磷解酶 澱粉 蔗糖
關鍵字(英)starch phosphorylase L-SP starch
摘要(中)澱粉是植物中含量最高的生物聚合物,也是人類所攝取的最主要多醣來源,因此釐清澱粉代謝的機制,將有助於作物改良與栽培,其長遠目標是解決糧食問題。但是植物澱粉代謝的機制非常複雜,探討這些代謝相關酵素的功能,主要是利用篩選植物的突變株,利用功能消失 (function disability) 與表現型變化 (phenotype change),來推論這些酵素在植物代謝上可能扮演之角色。以澱粉磷解? (starch phosphorylase,SP,EC 為例,為高等植物中參與澱粉代謝的酵素之一,其催化反應如下:?-glucose 1-phosphate + Glucan(n) ←→ Pi + Glucan(n+1)。但是由於突變株取得不易,至今對於 L-SP 的生理功能仍無法確定。植物澱粉的合成機制已經研究多年,一般認為主要參與合成澱粉的酵素是澱粉合成? (starch synthase,EC,此酵素以 ADP-Glc 或是 UDP-Glc 為葡萄糖單元體之來源,在短鏈糖引子 (sugar primer) 的非還原端 (non-reducing end) 加成延長糖鏈。分支酵素 (branch enzyme,EC 與去分支酵素 (debranching enzyme,EC,則可對直鏈糖分子進一步修飾,產生各種不同分支度的葡聚醣。由上述可知,澱粉合成?必須在糖引子存在下才能進行延長糖鏈反應。因此,最初糖引子的生成反應,應是後續澱粉高分子形成不可或缺的步驟,而具有合成最初糖引子活性的酵素,應該參與澱粉合成的最初反應。根據本實驗室先前的結果顯示,L-SP 在試管反應 (in vitro) 中具有 primer independent phosphorylase (PIPh) 活性,也就是合成糖引子之能力,因此,L-SP 可能在植物澱粉生合成初期,具有關鍵之貢獻。在 1996 年時 Moreno 發現,馬鈴薯原生質體內的 L-SP 蛋白質含量可利用蔗糖來誘導,在本論文發現其誘導表現的階層從基因的層次開始,蔗糖可以誘導 L-SP mRNA 表現量增加,進而使 L-SP 蛋白質與活性增加。蔗糖除了誘導 L-SP 表現之外,也誘導葉綠體中澱粉的累積,暗示了L-SP與澱粉合成之高度相關性。原預計使用原生質體配合配合上 RNA 干擾術,在細胞內 (in vivo) 系統,更進一步確認 L-SP 在植物澱粉合成之角色。但是由於培養原生質體不如預期簡單,故在這部份無法繼續下去。另一方面,在探討蔗糖誘導後葉片中澱粉代謝相關的系統生物學中發現,在基因層次上發現除了 L-SP 外,H-SP (H-from starch phosphorylase)、granule-bound starch synthase (GBSS) 以及 ADP-glucose pyrophosphatase (AGPase) 也隨著誘導時間增加而增加。而在蛋白質的層次上發現,隨著蔗糖誘導時間增加,Leucine aminopeptidase 表現量增加,但 Rubiso 以及 oxygen evolving protein 表現量卻下降。
摘要(英)Starch is the most abundant biopolymer in plants, and the major source of carbohydrate in human diet. Therefore, elucidation of the metabolism of starch biosyntheis and degradation will be a great mileston in crop breeding. In the long run, understanding the detailed mechanism might solve the problem of food crisis. However, the mechanism of starch metabolism is more than complicated. The major way to study starch metabolism would be the utilization of mutant that lose the function or alter the phenotype. For example, starch phosphorylase (EC, which catalyzes the reversible phosphorolysis of starch in higher plants, is the enzyme with obscure function. Due to the inability to obtain a mutant, the physiological role of SP remains uncler. It is generally accepted that starch biosynthesis is predominantly exerted by starch synthase (EC adding glucose unit to existing sugar primer by using ADG-Glc or UDP-Glc. The heterogeneity of starch is then complete with the assist of branching (EC and debrancing enzyme (EC Therefore, the formation of the sugar primer would be the key step for subsequent chain elongation is starch biosynthesis. On the other hand, enzymes that catalyze the formation of sugar primer should participate in the very initials stage in starch synthesis. Our previous study indicate that in vito L form SP (L-SP) catalyzed a primer independent amylase synthesizing activity, which means the ability to produce sugar primer. It implied that L-SP might have contribution at the early stage of starch biosynthesis. In 1996, it has been reported that the activity of L-SP in potato leaves increased when inducing by sucrose. In this study, it was also found that the induction by sucrose affected on transcriptional level. The mRNA, protein, and enzyme activity of L-SP all increased when sucrose was present. We also found that significant starch accumulation in the chloroplasts of potato leaves induced by sucrose. Many evidences support the idea that L-SP participate in starch biosynthesis. In order to validate this hypothesis, we have tried to use RNAi to knock down the expression of L-SP in the protoplast of potato leaves, and see if the loss of L-SP function in plant cells might hinder the starch accumulation by sucrose induction. Unfortunately, the culture and transfecton efficiency of protoplast was not as successful as expected. We also used 2-DE and realtime RT-PCR to evaluate the gene expression change in the potato leaves induced by sucrose. Interestingly, other than L-SP, the mRNA expression of H form starch phosphorylase (H-SP) and granule-bound starch synthase (GBSS) also increased in the sucrose induced leaves.On the level of protein expression, Leucine aminopeptidase was found increasing; Rubisco and oxygen evolving protein were found decreasing. The posssilbe mechanism is discussed.
論文目次目錄 I 謝誌 V 中文摘要 VII 英文摘要 VIII 第一章 概論 1 第二章 材料與方法 22 第三章 結果 60 第四章 圖表集 64 第五章 討論 84 參考資料 89 答問錄 95
參考文獻Al-Khalili, L., Cartee, G.D., and Krook, A. (2003). RNA interference-mediated reduction in GLUT1 inhibits serum-induced glucose transport in primary human skeletal muscle cells. Biochem Biophys Res Commun 307, 127-132. Andrews, A.T. (1986). Electrophoresis: Theory, techniques and biochemical and clinical application. Oxford university press. Ashrafi, K., Chang, F.Y., Watts, J.L., Fraser, A.G., Kamath, R.S., Ahringer, J., and Ruvkun, G. (2003). Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature 421, 268-272. Beckmann, C.O., and Roger, M. (1951). The question of the branching enzyme in potatoes. J Biol Chem 190, 467-480. Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366. Bolotina, T.T., and Petrova, A.N. (1953). [Phosphoglucomutase in potato tubers.]. Dokl Akad Nauk SSSR 88, 1027-1029. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254. Caplen, N.J., Parrish, S., Imani, F., Fire, A., and Morgan, R.A. (2001). Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A 98, 9742-9747. Chang, T.C., and Su, J.C. (1986). Starch Phosphorylase Inhibitor from Sweet Potato. Plant Physiol 80, 534-538. Chen, H.M., Chang, S.C., Wu, C.C., Cuo, T.S., Wu, J.S., and Juang, R.H. (2002). Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis. Physiol Plant 114, 506-515. Cogoni, C., Irelan, J.T., Schumacher, M., Schmidhauser, T.J., Selker, E.U., and Macino, G. (1996). Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. Embo J 15, 3153-3163. Cogoni, C., and Macino, G. (2000). Post-transcriptional gene silencing across kingdoms. Curr Opin Genet Dev 10, 638-643. de Fekete MAR, L.L., Cardini CE. (1960). Mechanism of starch biosynthesis. Nature 187, 918-919. Dunn, M.J. (1993). Gel Electrophoresis: proteins. Bios Scientific Publishers limited, Oxford, Unitied Kingdom. Dzitoyeva, S., Dimitrijevic, N., and Manev, H. (2001). Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system. Mol Psychiatry 6, 665-670. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498. Elbashir, S.M., Lendeckel, W., and Tuschl, T. (2001b). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15, 188-200. Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. (2001c). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 20, 6877-6888. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. Frydman, R.B., and Cardini, C.E. (1964). Biosynthesis of phytoglycogen from adenosine diphosphate D-glucose in sweet corn. Biochem Biophys Res Commun 14, 353-357. Gallo, R.C. (1971). Reverse transcriptase, the DNA polymerase of oncogenic RNA viruses. Nature 234, 194-198. Gerrits, N., Turk, S.C., van Dun, K.P., Hulleman, S.H., Visser, R.G., Weisbeek, P.J., and Smeekens, S.C. (2001). Sucrose metabolism in plastids. Plant Physiol 125, 926-934. Grishok, A., Pasquinelli, A.E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D.L., Fire, A., Ruvkun, G., and Mello, C.C. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23-34. Grishok, A., Tabara, H., and Mello, C.C. (2000). Genetic requirements for inheritance of RNAi in C. elegans. Science 287, 2494-2497. Grosser, J.W. (1994). In vitro culture of tropical fruits. In: Vasil, I. K. & Thorpe, T.A. (eds.). Plant Cell and Tissue Culture. Ch. 19, pp.475-496. Grosser, J.W., and Gmitter, F.G. Jr. (1990). Protoplast fusion and citrus improvement. Plant Breeding Reviews 8, 339-374. Guo, S., and Kemphues, K.J. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611-620. Gura, T. (2000). A silence that speaks volumes. Nature 404, 804-808. Hamilton, A.J., and Baulcombe, D.C. (1999). A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950-952. Hammond, S.M., Caudy, A.A., and Hannon, G.J. (2001). Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2, 110-119. Hans, C. (1940). The brankdown and synthesis of starch by enzyme system from pea seeds. Proc role Soc (London), B1280: 1421-1500. Hardy, E., and Castellanos-Serra, L.R. (2004). "Reverse-staining" of biomolecules in electrophoresis gels: analytical and micropreparative applications. Anal Biochem 328, 1-13. Hassid, W. (1962). The biosynthesis of polysaccharides from nucleoside diphosphate sugar. Biochem. Soc. Symp., 21:79-93. Herbers, K., Prat, S., and Willmitzer, L. (1994). Functional analysis of a leucine aminopeptidase from Solanum tuberosum L. Planta 194, 230-240. Hilson, P., Allemeersch, J., Altmann, T., Aubourg, S., Avon, A., Beynon, J., Bhalerao, R.P., Bitton, F., Caboche, M., Cannoot, B., Chardakov, V., Cognet-Holliger, C., Colot, V., Crowe, M., Darimont, C., Durinck, S., Eickhoff, H., de Longevialle, A.F., Farmer, E.E., Grant, M., Kuiper, M.T., Lehrach, H., Leon, C., Leyva, A., Lundeberg, J., Lurin, C., Moreau, Y., Nietfeld, W., Paz-Ares, J., Reymond, P., Rouze, P., Sandberg, G., Segura, M.D., Serizet, C., Tabrett, A., Taconnat, L., Thareau, V., Van Hummelen, P., Vercruysse, S., Vuylsteke, M., Weingartner, M., Weisbeek, P.J., Wirta, V., Wittink, F.R., Zabeau, M., and Small, I. (2004). Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. Genome Res 14, 2176-2189. Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E., and Prydz, H. (2002). Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res 30, 1757-1766. Hunter, C.P. (2000). Gene silencing: shrinking the black box of RNAi. Curr Biol 10, R137-140. Hutvagner, G., McLachlan, J., Pasquinelli, A.E., Balint, E., Tuschl, T., and Zamore, P.D. (2001). A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834-838. Hutvagner, G., and Zamore, P.D. (2002). RNAi: nature abhors a double-strand. Curr Opin Genet Dev 12, 225-232. Jorgensen, R.A., Cluster, P.D., English, J., Que, Q., and Napoli, C.A. (1996). Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol Biol 31, 957-973. Kamath, R.S., Martinez-Campos, M., Zipperlen, P., Fraser, A.G., and Ahringer, J. (2001). Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2, RESEARCH0002. Katome, T., Obata, T., Matsushima, R., Masuyama, N., Cantley, L.C., Gotoh, Y., Kishi, K., Shiota, H., and Ebina, Y. (2003). Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions. J Biol Chem 278, 28312-28323. Kerr, R.W. (1949). Action of beta amylase on amylose. Nature 164, 757. Ketting, R.F., Fischer, S.E., Bernstein, E., Sijen, T., Hannon, G.J., and Plasterk, R.H. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15, 2654-2659. La Cognata, U., Willmitzer, L., and Muller-Rober, B. (1995). Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase. Mol Gen Genet 246, 538-548. Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294, 853-858. Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. (2001). An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294, 858-862. Lee, E.Y., Marshall, J.J., and Whelan, W.J. (1971). The substrate specificity of amylopectin-debranching enzymes from sweet corn. Arch Biochem Biophys 143, 365-374. Lee, R.C., and Ambros, V. (2001). An extensive class of small RNAs in Caenorhabditis elegans. Science 294, 862-864. Lee, S.S., Lee, R.Y., Fraser, A.G., Kamath, R.S., Ahringer, J., and Ruvkun, G. (2003). A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet 33, 40-48. Lev?e V., B.M., Duval M., Bilodeau P., Aquin S.,V?zina L.-P. An efficient system for protoplast culture from alfalfa (Medicago sativa) suitable for plant transformation and regeneration. Medicago inc, 1020 Route de l’?glise, Bureau 600, Sainte-Foy, Qu?bec, Canada, G1V 3V9 418 658-9393 418 658-6699, www.medicago.com. Li, K., Lin, S.Y., Brunicardi, F.C., and Seu, P. (2003). Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma. Cancer Res 63, 3593-3597. Manche, L., Green, S.R., Schmedt, C., and Mathews, M.B. (1992). Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol 12, 5238-5248. Matzke, M.A., and Birchler, J.A. (2005). RNAi-mediated pathways in the nucleus. Nat Rev Genet 6, 24-35. Mello, C.C., and Conte, D., Jr. (2004). Revealing the world of RNA interference. Nature 431, 338-342. Minks, M.A., West, D.K., Benvin, S., and Baglioni, C. (1979). Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. J Biol Chem 254, 10180-10183. Moreno, S., and Tandecarz, J.S. (1996). Analysis of primer independent phosphorylase activity in potato plants: high levels of activity in sink organs and sucrose-dependent activity in cultured stem explants. Cell Mol Biol (Noisy-le-grand) 42, 637-643. Mori, H., Tanizawa, K., and Fukui, T. (1991). Potato tuber type H phosphorylase isozyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli. J Biol Chem 266, 18446-18453. Muda, M., Worby, C.A., Simonson-Leff, N., Clemens, J.C., and Dixon, J.E. (2002). Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochem J 366, 73-77. Napoli, C., Lemieux, C., and Jorgensen, R. (1990). Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289. Nelson, O.E., and Rines, H.W. (1962). The enzymatic deficiency in the waxy mutant of maize. Biochem Biophys Res Commun 9, 297-300. Nykanen, A., Haley, B., and Zamore, P.D. (2001). ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321. Paddison, P.J., Caudy, A.A., and Hannon, G.J. (2002). Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A 99, 1443-1448. Palauqui, J.C., Elmayan, T., Pollien, J.M., and Vaucheret, H. (1997). Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. Embo J 16, 4738-4745. Pothof, J., van Haaften, G., Thijssen, K., Kamath, R.S., Fraser, A.G., Ahringer, J., Plasterk, R.H., and Tijsterman, M. (2003). Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev 17, 443-448. Preiss, J., Okita, T.W., and Greenberg, E. (1980). Characterization of the Spinach Leaf Phosphorylases. Plant Physiol 66, 864-869. Rongine De Fekete, M.A., Leloir, L.F., and Cardini, C.E. (1960). Mechanism of starch biosynthesis. Nature 187, 918-919. Ruvkun, G. (2001). Molecular biology. Glimpses of a tiny RNA world. Science 294, 797-799. Sakurai, T., Satou, M., Akiyama, K., Iida, K., Seki, M., Kuromori, T., Ito, T., Konagaya, A., Toyoda, T., and Shinozaki, K. (2005). RARGE: a large-scale database of RIKEN Arabidopsis resources ranging from transcriptome to phenome. Nucleic Acids Res 33, D647-650. Saltman, P. (1953). Hexokinase in higher plants. J Biol Chem 200, 145-154. Schmid, A., Schindelholz, B., and Zinn, K. (2002). Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila. Trends Neurosci 25, 71-74. Sharp, P.A. (2001). RNA interference--2001. Genes Dev 15, 485-490. Sharp, P.A., and Zamore, P.D. (2000). Molecular biology. RNA interference. Science 287, 2431-2433. Smith, A.M., Zeeman, S.C., Thorneycroft, D., and Smith, S.M. (2003). Starch mobilization in leaves. J Exp Bot 54, 577-583. Steup, M. (1988). Starch degradation. In "The biochemistry of plants" Academia Press, New York, 14: 255-296. Tabara, H., Grishok, A., and Mello, C.C. (1998). RNAi in C. elegans: soaking in the genome sequence. Science 282, 430-431. Timmons, L., Court, D.L., and Fire, A. (2001). Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103-112. Timmons, L., and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395, 854. Trethewey RN, S.A. (2000). Starch metabolism in leaves. In: Leegood RC, Sharkey TD, von Caemmerer S, eds. Advances in photosynthesis Vol. 9. Photosynthesis: physiology and metabolism. Dordrecht: Kluwer Academic Publishers, 205-231. Worby, C.A., Simonson-Leff, N., and Dixon, J.E. (2001). RNA interference of gene expression (RNAi) in cultured Drosophila cells. Sci STKE 2001, PL1. Yang, S., Tutton, S., Pierce, E., and Yoon, K. (2001). Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol 21, 7807-7816. Yu, Y., Mu, H.H., Wasserman, B.P., and Carman, G.M. (2001). Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase. Plant Physiol 125, 351-359. Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33. Zeeman, S.C., Thorneycroft, D., Schupp, N., Chapple, A., Weck, M., Dunstan, H., Haldimann, P., Bechtold, N., Smith, A.M., and Smith, S.M. (2004). Plastidial alpha-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiol 135, 849-858. 陳翰民. (1997). 甘藷澱粉磷解?構造與功能之研究. 國立台灣大學農業化學所博士論文.
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