臺灣博碩士論文加值系統

蔗糖合成催化蔗糖與 UDP 形成果糖與 UDPG，後者即為多醣類生合成之前體。本論文之目的即為綠竹蔗糖合成基因的選殖與檢定。首先，以 l FIX II 為載體進行基因組庫之構築，接著利用含有植物蔗糖合成保守性序列的 RT-PCR 產物為探針，篩選綠竹基因組庫中的蔗糖合成基因，從 1.2 x 106 株選殖體中得到 25 株正反應株，再從中挑選八株進行限制分析與南方雜合反應後，挑選具有強烈雜合訊號的第 6 株選殖體進行次選殖與定序工作。
以 Not I 與 EcoR I 將第 6 株中長 13 kb 的插入片段 (命名為 I6) 水解為兩個片段，再分別將其次選殖到 pBluescript II SK 質體中，並加以定序。比對 I6 與 BSus cDNA 的序列發現，I6 包含一段與 BSus1b cDNA 具有高同質性的片段，此 7.6 kb 的 BSus1b 基因中共有 15 個 exons 與 14 個 introns，exon 序列與水稻 RSus1 具相當高的同質性，此外，在 BSus1b 基因的上游區域亦有發現 5 個 TATA box，和 2 個與蔗糖調控有關的序列。

Sucrose synthase catalyzes a reaction of sucrose and UDP to form fructose and UDPG, the latter being a precursor of complex saccharide biosynthesis. The objective of this thesis is to clone and characterize the gene encoding sucrose synthase in green bamboo Leleba oldhami. Genomic library was constructed using l FIX II vector and screened for sucrose synthase by using the RT-PCR product, which contain the conserved sequence of plant sucrose synthase, as a probe. There were 25 positive clones screened from 1.2 x 106 recombinant clones. Eight clones were selected randomly and analyzed by restriction enzymes and Southern hybridization. The No.6 clone with the highest hybridization signal was selected for further analysis.
The 13 kb insert of No.6 clone, named I6, was digested by NotI and EcoRI into two fragments. They were individually subcloned into plasmid pBluescript II SK and were subjected to sequencing. By comparing the sequence of I6 with those of BSus cDNAs, we found that I6 contained a Sus sequence with highest identity to BSus1b cDNA. There were fifteen exons and fourteen introns in this 7.6 kb BSus1b gene, which was highly homologuos to the RSus1 gene. Furthermore, five TATA boxes and two sucrose response elements sequence were found in the upstream region of the BSus1b gene.

目錄
目錄I
縮寫表 IV
中文摘要 VI
Abstract VII
第一章 緒論1
1.1 蔗糖及蔗糖合成1
1.2 植物蔗糖合成基因的研究狀況3
1.3 概說竹筍4
1.4 本論文之緣起與研究目的5
第二章 材料與方法7
2.1 實驗材料7
2.2 藥品與儀器7
2.2.1 藥品 7
2.2.2 儀器8
2.3 實驗方法9
2.3.1 基因組庫 (genomic library) 的建構9
2.3.1.1竹筍基因組DNA (genomic DNA) 之抽取9
2.3.1.2 DNA之電泳分析法10
2.3.1.2基因組DNA Sau3AI限制部分切割片段之製備11
A Sau3AI最適作用條件之探討11
B. 大量 DNA 之 Sau3AI 部份切割11
2.3.1.3基因組DNA片段之部分填補12
2.3.1.4將基因組DNA片段與載體 (l FIX II) 接合12
2.3.1.5試管外噬菌體的包裝13
2.3.1.6噬菌體價數的測定13
2.3.2 蔗糖合成基因的選殖14
2.3.2.1反轉錄-聚合連鎖反應14
A.第一股cDNA的合成14
B.聚合連鎖反應15
2.3.2.2 選殖體之篩選及確認 15
A.基因組庫之塗佈與轉印15
B.探針之製備16
C.探針雜合反應16
D.正反應選殖株之分離純化17
2.3.2.3 噬菌體 DNA 之抽取17
A.噬菌體之培養17
B.噬菌體的純化及其DNA抽取18
2.3.2.4 DNA片段的分離與純18
A. DEAE membrane吸附法18
B. Silica gel 吸附法19
2.3.2.5 Southern 轉印法20
2.3.2.6質體的轉形21
2.3.2.7重組質體之快速檢定22
2.3.2.8質體小量分離法22
2.3.2.9 DNA 序列縮減法 (Nested deletion)23
A.質體之限制切割23
B. 5’ 端突出之填補保護23
C. Exonuclease III 對DNA之水解24
2.3.2.10 DNA定序25
2.3.2.11序列分析25
第三章 結果與討論26
3.1 基因庫的建構26
3.1.1 綠竹筍 DNA 抽取結果26
3.1.2 Sau3AI 部分水解片段之製備26
3.1.3 基因組庫建構結果27
3.2 蔗糖合成基因的選殖與檢定28
3.2.1正反應株的性質鑑定28
3.2.2第六株選殖體之次選殖30
3.2.3 DNA 序列縮減法30
3.2.4定序與序列比對32
第四章 結論於未來展望35
參考文獻36
圖與表41

Alwine, J.C., Kemp, D.J. and Stark, G.R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U.S.A 74, 5350-5354
Ausubel, F. M., Brent, R., Kingston, R.E., Moore, D.D.,Seidman, J.G., Smith, J.A. and Struhl, K. (1987). Current Protocol in Molecular Biology. John Wiley and Sons, Inc, New York.
Cardini, C.E., Leloir, L.F. and Chiriboga, J. (1995). The biosynthesis of sucrose. J. Biol. Chem. 214, 149-155.
Chan, H.Y., Ling, T.Y., Juang, R.H., Ting, I.N., Sung, H.Y. and Su, J.C. (1990). Sucrose synthase in rice plants. Plant Physiol. 94, 1456-1461.
Chengappa, S., Guilleroux, M., Phillips, W. and Shields, R. (1999). Transgenic tomato plants with decreased sucrose synthase are unaltered in starch and sugar accumulation in the fruit. Plant Mol. Biol. 40, 213-221.
Chopra, S., Del favero, J., Dolferus, R. and Jacobs, M. (1992). Sucrose synthase of Arabidopsis : genomic cloning and sequence characterization. Plant Mol. Biol. 18, 131-134.
Chourey, P.S. and Nelson, O.E. (1976). The enzymatic deficiency conditioned by the shrunken-1 mutation in maize. Biochem. Genet. 14, 1041-1055.
Chourey, P.S., Taliercio, E.W. and Kane, E.J. (1991). Tissue-specific expression and anaerobically induced post-transcripitional modulation of sucrose synthase gene in Sorghum bicolor M. Plant Physiol. 96, 485-490.
Cross, K.C. and Pharr, D.M. (1982). Cucumber fruit sucrose synthase isozymes. Phytochemistry. 21, 1241-1244.
Echt, C.S. and Chourey, P.S. (1985). A comparison of two sucrose synthetase isozymes from normal and shrunken-1 maize. Plant Physiol. 79, 530-536.
Fu, H., Kim, S.Y. and Park, W.D. (1995). A potato Sus3 sucrose synthase gene contains a context-dependent 3'' element and a leader intron with both positive and negative tissue- specific effects. Plant Cell 7, 1395-1403.
Fu, H., Kim, S.Y. and Park, W.D. (1995). High-level tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5'' and 3'' flanking sequences and the leader intron. Plant Cell 7, 1387-1394.
Geigenberger, P. and Stitt, M. (1993). Sucrose synthase catalyses a readily reversible reaction in vivo in developing potato tubers and other plant tissues. Planta 189, 329-339.
Grierson, C., Du, J.S., Zabala, M. de T., Beggs, K., Smith, C., Holdsworth, M. and Bevan, M. (1994). Separate cis sequences and trans factors direct metabolic and developmental regulation of a potato tuber storage protein gene. Plant J. 5, 815-826.
Hisajima, S. and Ito, T. (1981). Purification and properties of sucrose synthase from morning-glory callus cells. Biol. Plant. 23, 356-364.
Huang, D.Y. and Wang, A.Y. (1998). Purification and characterization of sucrose synthase isozymes from etiolated rice seedlings. Biochem. Mol. Biol. Int. 46, 107-113.
Maas, C., Laufs, J., Grant, S., Korfhage, C. and Werr, W. (1991). The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold. Plant Mol. Biol. 16, 199-207.
Marana, C., Garcia-Olmedo, F. and Carbonero, P. (1988). Linked sucrose synthase genes in group-7 chromosomes in hexaploid wheat (Triticum aestivum L.). Gene 63, 253-260.
Marana, C., Garcia-Olmedo, F. and Carbonero, P. (1990). Differential expression of two types of sucrose synthase-encoding genes in wheat in response to anaerobiosis, cold shock and light. Gene 88, 167-172.
Murray, M. G. and Thompson, W. F. (1980). Rapid isolation of high-molecular-weight plant DNA. Nucl. Acids Res. 8, 4321-4325.
Nguyen-Quoc, B., Krivitzky, M., Huber, S.C. and Lecharny, A. (1990). Sucrose synthase in developing maize leaves. Plant Physiol. 94, 516-523.
Salanoubat, M., Belliard, G. (1987). Molecular cloning and sequencing of sucrose synthase cDNA from potato (Solanum tuberosum L.): preliminary characterization of sucrose synthase mRNA distribution. Gene 60, 47-56
Salnikov, V.V., Grimson, M.J., Delmer, D.P. and Haigler, C.H. (2001). Sucrose synthase localizes to cellulose synthesis sites in tracheary elements. Phytochemistry 57, 823-833.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, ed 2, Cold Spring Harbor Press, New York
Schaffer, A.A., Aloni., B. and Fogelman. E. (1987). Sucrose metabolism and accumulation in developing fruit of cucumis. Phytochemistry 26, 1883-1887.
Seed, B. (1982). Diazotizable acrylamine cellulose papers for the coupling and hybridization of nucleic acids. Nucl. Acids Res. 10, 1799-1810.
Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.
Sturm, A., Lienhard, S., Schatt, S. and Hardegger, M. (1999). Tissue-specific expression of two genes for sucrose synthase in carrot (Daucus carota L.). Plant Mol. Biol. 39, 349-360.
Su, J.C. (1965) Carbohydrate metabolism in the shoots of bamboo Leleba oldhami. I Preliminary survey of soluble saccharides and sucrose-degrading enzymes. Bot. Bull. Acad. Sinica, 6, 153-159.
Su, J.C. and Preiss, J. (1978). Purification and properties of sucrose synthase from maize kernels. Plant Physiol. 61, 389-393.
Su, J.C., Wu, J.L. and Yang, C.L. (1977). Purification and characterization of sucrose synthetase from the shoot of bamboo Leleba oldhami. Plant Physiol. 60, 17-21.
Tang, G.Q. and Sturm, A. (1999). Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning. Plant Mol. Biol. 41, 465-479
Vande Woude, G.F., Oskarsson, M., Enquist, L.W., Nomura, S., Sullivan, M. and Fischinger, P.J. (1979). Cloning of integrated Moloney sarcoma proviral DNA sequences in bacteriophage l. Proc. Natl. Acad. Sci. 76:4464.
Vasil, V., Clancy, M., Ferl, R. J., Vasil, I. K. and Hannah, L. C. (1989). Increase gene expression by first intron of maize shrunken-1 locus in grass species. Plant Physiol. 91, 1575-1579.
Wang, A.Y., Yu, W.P., Juang, R.H., Huang, J.W., Sung, H.Y. and Su, J.C. (1992). Presence of three rice sucrose synthase genes as revealed by cloning and sequencing of cDNA. Plant Mol. Biol. 18, 1191-1194.
Wang, F., Smith, A.G., Brenner, M.L. (1993). Isolation and sequencing of tomato fruit sucrose synthase cDNA. Plant Physiol. 103, 1463-1464.
Weber, H., Borisjuk, L., Heim, U., Sauer, N. and Wobus, U. (1997). A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seed. Plant Cell 7, 1835-1846.
Wolosiuk, R.A. and Pontis, H.G. (1971). Evidence of the existence of two forms of sucrose synthetase. FEBS Lett. 16, 237-240.
Yeh, D.B. and Su, J.C. (1972). Purification and charaterization of sucrose synthetase form banana fruits. J. Chin. Biochem. Soc. 1, 37-52.
Zrenner, R., Salanoubat, M., Willmitzer, L. and Sonnewald, U. (1995). Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant J. 7, 97-107.
王素秋 (1989) 豌豆蔗糖合成之研究, 碩士論文, 國立台灣大學農業化學系.
宋賢一 (1973) 蔗糖合成酵素在植物醣類代謝之功用, 博士論文, 國立台灣大學農業化學系.
岩素芬 (1992) 水稻蔗糖合成異構之純化與鑑定, 博士論文, 國立台灣大學農業化學系.
林秋宏 (2001) 綠竹筍蔗糖合成 cDNA 之選殖與檢定, 碩士論文, 國立台灣大學農業化學系.
蘇佑倫 (1999) 水稻蔗糖合成 RSus2 基因之 intron1 及 exon1 對基因表現之影響, 碩士論文, 國立台灣大學農業化學系.