臺灣博碩士論文加值系統

摘 要
蔗糖合成催化蔗糖與UDP形成果糖與UDPG的反應，後者即為多醣類生合成之前體。為了了解蔗糖合成在快速生長的竹筍中扮演的角色，因此本論文之目的即為進行綠竹筍蔗糖合成cDNA的選殖與檢定。以含有植物蔗糖合成 保守性序列的RT-PCR產物為探針，篩選綠竹筍cDNA庫，得到四種不同型式的蔗糖合成cDNA，分別為BSus1a、BSus1b、BSus2a以及BSus2b。BSus1a、BSus1b cDNA推衍出的胺基酸序列皆為816個胺基酸，分子量分別為92.83、92.98 kDa；與其它植物之Sus1-type蔗糖合成胺基酸序列的同質性最高，為90~96%之間。BSus2a及BSus2b cDNA推衍出的胺基酸序列皆為808個胺基酸，分子量分別為92.25、92.12 kDa；與Sus2-type蔗糖合成胺基酸序列的同質性最高，為89~92%之間。BSus1a與BSus1b mRNA在葉中的含量高於竹筍 (shoots)，而BSus2a及BSus2b mRNA則主要存在於竹筍中。以pTrcHisA為表現載體，將四種BSus cDNA胺基酸轉譯區的序列轉形至大腸桿菌TOP10中表現。得到的重組蛋白質具有蔗糖合成的酵素活性，可被抗水稻蔗糖合成之多源性抗體辨認。由上述之結果證實了四種不同型式的cDNA，確實為綠竹筍蔗糖合成之cDNA。

Abstract
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 cDNA encoding sucrose synthase in the shoots of bamboo in order to understand the roles played by sucrose synthase in this rapid-growing plant. A cDNA library constructed from poly(A)+RNA of green bamboo shoots was screened for sucrose synthase by using the RT-PCR product, containing the conserved sequence of plant sucrose synthase, as a probe. Four different types of sucrose synthase cDNA, BSus1a, BSus1b, BSus2a and BSus2b were identified. BSus1a and BSus1b both encode polypeptides of 816 amino acids with relative molecular masses of 92.83 and 92.98 kDa and have highest amino acid sequence homology with Sus1-type sucrose synthase of other plants (90~96% identity). BSus2a and BSus2b both encode polypeptides of 808 amino acids with relative molecular masses of 92.25 and 92.12 kDa and have highest amino acid sequence homology with Sus2-type sucrose synthase (89~92% identity). BSus1a and BSus1b mRNA were detected at higher level in leaves than in shoots while BSus2a and BSus2b mRNA were detected predominantly in shoots. The coding region of each BSus cDNA was subcloned into an expression vector, pTrcHis A. The constructed expression plasmids were then transformed into E.coli TOP10 for expression. The recombinant proteins possessed sucrose synthase activity and showed cross-reaction to a polyclonal antibody against rice sucrose synthase. These results prove conclusively that four cDNAs encode sucrose synthase in green bamboo shoots.

目 錄
目錄……………………………………………………………………………Ⅰ
縮寫表………………………………………………………………………..Ⅲ
摘要…………………………………………………………………………..Ⅴ
Abstract…………………………………………………………………….Ⅵ
第一章 緒論………………………………………………………………….1
1 蔗糖合成之生化特性及生理角色……………………………………..1
1.1 蔗糖合成之生化特性……………………………………………..1
1.2 蔗糖合成之生理角色……………………………………………..3
1.2.1 蔗糖合成在澱粉合成上扮演之角色……………………..3
1.2.2 蔗糖合成在纖維素合成上扮演之角色…………………..4
1.2.3 蔗糖合成可能扮演之其它角色…………………………..5
2 蔗糖合成之基因與基因表現…………………………………………..6
3 本實驗室在水稻蔗糖合成的研究基礎………………………………..8
4 本論文之研究目的與方向………………………………………………..9
4.1 研究動機與目的………………………………………………………9
4.2 本論文之研究重點……………………………………………….11
第二章 材料與方法…………………………………………………………12
1 實驗材料料………………………………………………………………12
1.1 植物材料…………………………………………………………….12
1.2 載體………………………………………………………………….12
1.3 大腸桿菌菌株…………………………………………………….12
2 實驗藥品…………………………………………………………………13
2.1 一般化學試劑……………………………………………………….13
2.2 限制及核酸修飾酵素…………………………………………….13
2.3 培養基……………………………………………………………….13
2.4 放射性藥品………………………………………………………….13
3 重要儀器設備……………………………………………………………14
4 實驗方法…………………………………………………………………15
4.1 RNA之抽取及分析方法…………………………………………….15
4.2 DNA之抽取及分析方法…………………………………………….20
4.3 cDNA的合成………………………………………………………...24
4.4 cDNA庫的建立……………………………………………………...26
4.5 cDNA庫之篩選……………………………………………………...28
4.6 表現載體之建構…………………………………………………….30
4.7 重組蛋白質之檢定…………………………………………………32
4.8 放射性探針之製備…………………………………………………37
4.9 其他重要實驗方法…………………………………………………39
4.10 序列分析…………………………………………………………..44
第三章 結果與討論………………………………………………………...45
1 綠竹筍RNA的抽取……………………………………………………..45
1.1 RNA之抽取與poly(A)+RNA之純化…………………………..45
1.2 RNA之完整性分析…………………………………………...45
2 以RT-PCR進行綠竹筍蔗糖合成cDNA之選殖……………………….46
2.1 部分Sus cDNA之選殖……………………………………………...46
2.2 5''端與3''端片段之選殖………………………………….……...47
2.3 RT-PCR選殖方法之檢討……………………………………………49
3 全長Sus cDNA之選殖…………………………………………………..49
3.1綠竹筍cDNA庫的建立……………………………………………..49
3.2 cDNA庫之篩選…………...…………………………………………50
3.3正反應株的鑑定……………………………………………………..50
4 Sus cDNA序列分析……………………………………………………...51
4.1 cDNA序列及其推衍出之胺基酸序列……………………………...51
4.2 綠竹筍之Sus異構基因……………………………………………..53
4.3 3''端與5''端不轉譯區…………………………………………....54
4.4 與RT-PCR結果之比對……………………………………………..55
5 綠竹筍SuS基因表現與拷貝數目的探討………………………………55
5.1 專一性探針的製備………………………………………………….55
5.2 綠竹筍不同組織蔗糖合成基因表現的探討…………………….56
5.3 BSus異構基因之表現與異構之關係…………………………….57
5.4綠竹筍蔗糖合成基因拷貝數目分析……………………………..58
6 重組蛋白質之檢定………………………………………………………59
6.1 表現載體之建構…………………………………………………….59
6.2 重組蛋白質表現之探討…………………………………………….60
第四章 結論與展望………………………………………………………...62
參考文獻…………………………………………………………………….65
圖與表……………………………………………………………………….72

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