臺灣博碩士論文加值系統

中文摘要
蔗糖磷酯合成（Sucrose phosphate synthase，簡稱 SPS）是植物蔗糖生合成的一個重要酵素。以台農57號甘藷塊根為材料，粗抽液經 DEAE-Sephacel 離子交換層析，24 % PEG沉澱，w-aminohexyl Sepharose 4B和FPLC Mono-Q管柱層析進行酵素純化，得到純化倍率約64倍，回收率約 9% 的SPS製備液。經原態 PAGE 分析測得酵素分子量約 540 kDa，另以SDS-PAGE分析推測其單元體分子量約為130~140 kDa，得知 SPS 可能由同質四元體組成。且其pI值約5.29。
甘藷塊根 SPS並不會受G6P及Pi的異位調控，與光合組織所得者不同。而ATP, ADP, AMP, UTP, UDP, UMP會降低 SPS的活性約 40-50%。但 CTP, GTP, GDP對SPS的活性並沒有產生影響。
甘藷塊根 SPS 對於硫氫化合物很敏感，在低濃度 (0.1 mM) 時 —SH 化合物對酵素有安定作用，但過高 (10 mM 以上) 則反而有明顯抑制作用。SPS 在葡萄糖，葡萄糖胺，麥芽糖，和乳糖中活性會增加，然而 SPS 則被 d-葡萄糖酸內酯完全抑制，可能是酵素會形成不穩定的葡萄糖-酵素化合物之故。硫氫化合物之抑制劑 PCMBS 會隨著濃度增加而降低 SPS 的活性。而另一抑制劑 cibacron blue F3G-A 為 UDPG 的類似物，可以對 SPS 的 UDPG 結合區產生抑制。
以抑制劑 DEP (diethyl pyrocarbonate) 對SPS作修飾作用的分析，因其可以修飾蛋白質中的組胺酸而抑制SPS 催化反應。且SPS 的活性會隨著DEP濃度的增加而減少。且DEP抑制 SPS 的速率常數為 0.25 mM-1. min-1和約有0.62個 DEP可和一個 SPS 分子結合。當僅加入受質 UDPG 或 Ｆ6-P 時， SPS活性會被DEP抑制。但若同時加入UDPG和　F 6-P 時，DEP則完全不能抑制 SPS的活性。表示DEP 修飾作用位置應該在UDPG 結合區附近，
利用合成胜片段來製造抗體：其胺酸序列為IRGENMELGRDSDTG，與本實驗所篩選出的甘藷葉子 SPS cDNA推導之胺酸序列中，第177~191 個胺酸完全相同。製備之抗體血清對抗原 SPS之效價測定，當稀釋至 2000倍時免疫呈色反應仍很強，且加入抗體可抑制 SPS 活性。以10% SDS-PAGE 進行免疫分析，約可在130 kDa 染出一條色帶，此色帶應為SPS，但另有一條色帶出現約為 70 kDa，此色帶可能是SPS 的降解產物。以免疫呈色法分析甘藷不同部位組織的 SPS 含量，發現以葉子含量最多，塊根含量最少；而葉子中以幼嫩葉子中 SPS含量較少，成熟的葉子較多。由SPS 的降解研究發現在約3小時 SPS 開始被降解，而在 24小時後 SPS 被完全降解。而加入蛋白抑制劑MG-132時，SPS 並不會被降解，表示用以降解甘藷 SPS之蛋白可能是屬於半胱胺酸型蛋白。且利用免疫沉澱法分析SPS在免疫呈色上可以染出三條色帶分別為 130、100及85 kD，而其中130 kD 為 SPS次單元體，100 kD及85 kD 可能為SPS的降解蛋白片段。
以中型甘藷塊根和葉子的 total RNA 為模版，利用 RT-PCR 可合成出 900 bp 和 600 bp cDNA。經核酸定序的比對證實其為 SPS (GenBank accession no. AF135800, GenBank accession no. AY007199)，其推導出之胺酸序列和其他植物比較有相當高的相似性。就篩選出來的甘藷塊根與葉子 cDNA 序列比較有 120 bp 的重疊，其推導出之胺酸相似性為 90%，差異的部分均為非保守性的胺酸。在北方點墨法分析發現甘藷塊根內的 SPS 含量很少，葉子含量最多。在葉子中年輕葉子內的 SPS 含量較少，成熟葉子含量較多。以南方點墨法分析發現甘藷內 SPS 基因在體染色體的 copy number 為一倍。
甘藷 SPS 以免疫組織染色分析發現，在塊根中 SPS 的分布主要在脈狀形成層 (vascular cambium ，VC) 、導管 (xylem vessels， XV) 的周圍和澱粉粒 (starch granule ，SG)的周圍。在甘藷蔓莖中，主要存在的部分亦為脈狀形成層，但乳汁管 (laticifer)中也有發現。而甘藷葉子中，分布的位置在葉肉細胞 (mesophyll cell， M)、葉鞘細胞 (bundle-sheath cell， BS) 附近和導管的周圍。經原位雜交分析發現，甘藷塊根中 SPS 的分布主要在脈狀形成層和澱粉粒的周圍有明顯的呈色。而在甘藷蔓莖中，主要存在的部分亦為脈狀形成層。但在甘藷葉子中，則僅葉肉細胞的周圍有明顯的呈色。

Abstract
Sucrose phosphate synthase (SPS) is one of the key enzymes in the sucrose biosynthesis pathway. SPS was purified 40 folds from crude extract of sweet potato tuberous roots by the methods of batch elution from DEAE-Sephacel, PEG precipitation, w-aminohexyl Sepharose 4B affinity and Mono Q anion exchange chomatographies. Purified SDS was estimated to be 540 kDa from a native PAGE and 130~140 kDa from a SDS-PAGE, respectively. Therefore SPS was predicted to be a homotetramer in its native form.
SPS from the sweet potato tuberous root, which is different from those of photosynthetic tissues, is not allosterically regulated by glucose 6-phosphate and inorganic phosphate. The nucleotides AMP, ADP, ATP, UMP, UDP, UTP and TDP inhibit the enzyme activities about 30~50%. The enzyme was sensitive to sulfydryl reagents, but its activity can be restored with dithiothreitol orβ-mercaptoethanol. The enzyme was activated by glucose, glucosamine, maltose, lactose, while inhibited by
Chemical modification of SPS by diethyl pyrocarbonate (DEP), which modify the histidyl residues of the enzyme protein, resulted in the inactivation of the enzyme activity. The inactivation was dependent on the concentration of the modifying reagent and the time of incubation. The inhibition of SPS by DEP in the rate constants was 0.25 mM-1 min-1 . An inactive enzyme-inhibitor complex was shown to be composed of 1 (enzyme)：0.62 (inhibitor) using kinetics experiments. UDPG and F6P, substrates of SPS, protected the enzyme against the inactivation by the modifying reagents. This suggested that the histidine residues may be involved in the binding of these substrates and are essential for the catalytic activity. The results suggest the presence of histidine residue at or near the active site of the enzyme.
Polyclonal antibody against a synthetic peptide (IRGENMELGRDSDTG) was raised rabbit, which corresponded to the 177~191 amino acid sequence of SPS from sweet potato leaf. The serum titer titration can be diluted to two thousand folds and still show satisfying potency. The antibody could inhibit the SPS activity dramatically. Two protein bands with 130 and 70 kDa, respectively, were detected using Western blot. The 70 kDa cound be one of degraded fragments of SPS. Elevated level expression of SPS protein was observed in leaf using Western blot, while a lower expression was seen in tuberous root. Furthermore, the higher amounts of SPS expression were observed in mature leaf than in young leaf of sweet potato.
Using immunoprecipitation techniques, it was found that the antibody could bind 3 peptide fragments. The 130 kDa fragment is the SPS subunit. The 100 and 85 kDa fragments are very likely to be the degraded SPS consisting the epitope.
Two partial cDNA of SPS, with 900 bp and 600 bp cDNA respectively (GenBank accession no. AF135800, GenBank accession no. AY007199) were amplified by RT-PCR using sweet potato tuberous root and leaf total RNA as template. The two cDNA fragments were cloned and were used to study expression of the enzyme during tuberous root and leaf development. The deduced amino acid sequences from determined sequences show that sweet potato SPS has a high homology to the SPS from other species. The cDNA between tuberous root and leaf were overlapped by 120 bp. Within the 120 bp, the amino acid sequences from tuberous root and leaf share about 90% identity. The SPS trascripts could be detected in tuberous root, stem and leaf tissues examined albeit at varying levels. Elevated level of SPS transcript was observed in leaf, while a lower expression was seen in tuberous root. From the RNA blot analysis of young and mature leaves, it was found higher amount of SPS transcripts were observed in mature leaf. And from the results of genomic Southern blot analysis, it was suggested that SPS is an one-copy number gene.
Immunohistological analyses for SPS show that the protein is localized in vascular cambium , xylem vessels, starch granule, bundle-sheath cells and mesophyll cells of sweet potato. In situ hybridization analysis for SPS showed that the protein is localized in vascular cambium, starch granule, and mesophyll cells of sweet potato tissues.

封面
目錄
縮寫表
中文摘要
英文摘要
第一章緒論
1.1蔗糖在植物體內的生理功能
1.2蔗糖磷酯合成?
1.3蔗糖磷酯脢活性之調控
1.4蛋白質14-3-3調控蔗糖磷酯合成脢活性
1.5不同植物間蔗糖磷酯合成脢之比較
1.6蔗糖磷酯合成脢之分子生物學研究
1.7蔗糖磷酯合成脢可能出現複合體
1.8蔗糖磷酯合成脢基因在轉殖植株表現之研究
1.9蔗糖磷酯合成脢在細胞內的角色
1.10蔗糖磷酯合成脢之其他研究
第二章實驗緣起
第三章材料與方法
3.1實驗材料
3.2蛋白質定量法
3.3酵素活性分析
3.3-1蔗糖磷酯合成脢之 酮反應法
3.3-2蔗糖合成脢之純化
3.4蔗糖磷酯合成脢之純化
3.4-1酵素的粗抽及PEG分割
3.4-2w-aminohexyl-Sepharose 4B管柱層析
3.4-3快速蛋白質液相層析
3.4-4膠體過濾管柱
3.5一般電泳檢定法
3.5-1原態膠體電泳
3.5-2SDS膠體電泳
3.5-3膠體染色法：CBR蛋白質染色法
3.5-4膠片乾燥
3.5-5等電交集法
3.6SPS專一性抗體之製備
3.7酵素免疫分析法
3.8蛋白質免疫轉印法
3.8-1蛋白質電泳轉印法
3.8-2酵素免疫染色
3.8-3免疫沉澱法
3.9RNA和DNA之抽取與分析方法
3.9-1全部RNA之抽取
3.9-2RNA甲醛洋菜膠體電泳分析法
3.9-3植物組織DNA的抽取
3.10質體DNA之抽取
3.10-1快速篩選法
3.10-2小量抽取(Miniprep)
3.10-3聚合?鏈鎖反應
3.10-4RT-PCR(reverse transcriptase-PCR)
3.10-5DNA洋菜膠體電泳法
3.10-6DNA片段之分離純化
3.10-7質體DNA之切除
3.10-8pGEMR-T easy vector與insert之粘接
3.10-9質體之轉型
3.10-10質體轉移至E.Coli JM109
3.11核酸轉印
3.11-1DNA轉印法(Southern blot)
3.11-2DNA轉印
3.11-3RNA轉印法(Northern blot)
3.11-4雜交反應
3.11-5探針反應
3.12甘藷蔗糖磷酯合成脢（SPS）cDNA專一性探針(probe)的製備
3.12.1甘藷塊根SPScDNA專一性探針的製備
3.12.2甘藷葉子SPScDNA專一性探針的製備
3.13DNA定序
3.14植物組織切片的製備
3.15原位雜交(In situ hybridization)
3.15-1DIG-labeled RNA探針之製備
3.15-2In situ hybiridization
3.15-3酵素免疫染色法
3.15-4PAS染色法
3.15-5脫水與封片
第四章結果與討論
4.1甘藷塊根蔗糖磷酯合成脢之純化與性質研究
4.1-1甘藷不同部位組織蔗糖磷酯合成之活性大小
4.1-2甘藷塊根蔗糖磷酯合成脢蛋白質的純化
4.1-3甘藷塊根蔗糖磷酯合成脢之分子量測定
4.1-4甘藷塊根蔗糖磷酯合成脢之分子等電泳分析
4.1-5核?酸對甘藷塊根蔗糖磷酯合成脢之活性影響
4.1-6醣類對甘藷塊根蔗糖磷酯合成脢之活性影響
4.1-7硫氫化合物對甘藷塊根蔗糖磷酯合成脢之活性影響
4.1-8抑制劑對甘藷塊根蔗糖磷酯合成脢之活性影響
4.2DEP(diethyl pyrocarbonate)對於甘藷塊根蔗糖磷酯合成脢修飾作用的分析
4.2-1不同DEP的濃度和時間對於蔗糖磷酯合成脢修飾作用的影響
4.2-2DEP對蔗糖磷酯合成脢的影響
4.2-3NH2OH減緩DEP對蔗糖磷酯合成脢的活性抑制
4.2-4DEP對於蔗糖磷酯合成脢中加入DTT和DTNB修飾作用的影響
4.2-5DEP的修飾作用對於蔗糖磷酯合成脢中受質和活化劑的影響
4.2-6甘藷蔗糖磷酯合成脢可能和DEP作用的胺酸位置圖
4.3SPS專一性抗體之製備
4.3-1搞原決定基區域勝?片段的選擇和抗體的製備
4.3-2測量抗體對抗原SPS之效價測定
4.3-3抗體性質的分析
4.3-4甘藷不同部位組織中SPS的活性
4.3-5甘藷塊根及葉子組織中SPS的降解研究
4.3-6利用免疫沉澱法分析甘藷蔗糖磷酯合成
4.4甘藷塊根及葉子分子生物學研究
4.4-1甘藷塊根及葉子總RNA的抽取
4.4-2甘藷塊根蔗糖磷酯合成脢基因之選殖
4.4-3甘藷葉子蔗糖磷酯合成脢基因之選殖
4.4-4甘藷塊根及葉子蔗糖磷酯合成脢cDNA及其胺酸的比對
4.4-5甘藷塊根及葉子蔗糖磷酯合成脢核酸探針的製備
4.4-6北方點墨法分析
4.4-7南方點墨法分析
4.5甘藷內蔗糖磷酯合成脢組織定位的分析
4.5-1甘藷組織內PAS染色分析
4.5-2甘藷組織蔗糖磷酯合成脢的免疫組織染色分析
4.5-3甘藷組織蔗糖磷酯合成脢的原位雜交分析
第五章結論與展望
第六章參考文獻

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