臺灣博碩士論文加值系統

蔗糖合成酶催化蔗糖及 UDP 轉換為果糖及 UDPG 的可逆反應。在目前已知的六種水稻蔗糖合成酶異構基因 (RSus) 中，RSus1 及 RSus3 已被轉殖至酵母菌 Pichia pastoris 並表現重組 RSuS 蛋白質。然而核苷酸定序結果顯示位於 P. pastoris 表現質體中的 RSus1 及 RSus3 cDNA 皆帶有突變。本研究建構帶有正確 RSus1 及 RSus3 ORF 序列之表現質體，並成功以 P. pastoris 表現重組野生型 RSuS1 及 RSuS3 蛋白質，同時兩重組蛋白質皆以 DEAE-Sephacel 離子交換層析、 Sephacryl S-300 膠體過濾層析及 Resource-Q 陰離子交換快速液相層析進行純化。
重組野生型 RSuS3 蛋白質的蔗糖分解與合成方向比活性皆比突變型 RSuS3 蛋白質 (mRSuS3) 高出許多，同時兩者間蔗糖分解與合成方向比活性之比值也有明顯差異。由酵素動力學的分析結果，比較對 UDPG 的親和力，重組野生型 RSuS3遠高於重組 mRSuS3。這些分析結果顯示位於 mRSuS3 上的變異，包括 T4P、V39A、D129G 及 F680S，在 RSuS3 與 UDPG 的結合上，以及 RSuS3 在蔗糖合成及分解方向的活性影響上，皆扮演著重要的角色。
由酵素動力學的研究結果亦發現，重組 RSuS1 對 UDPG 及果糖的親和力遠高於 mRSuS1；在蔗糖合成方向比活性的比較上，重組 RSuS1 亦高於 mRSuS1。重組 RSuS1 與 mRSuS1 之 C 端序列的差異可能為影響活性及與基質結合的重要原因。

Sucrose synthase (SuS) catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. Among six known rice Sus genes (RSus), RSus1 and RSus3 were previously transformed into yeast Pichia pastoris and the recombinant RSuS proteins were expressed successfully. However, the results of nucleotide sequencing of expression plasmids containing RSuS1 or RSuS3 cDNA showed that there were mutations in both RSus1 and RSus3. In this research, the expression plasmids carrying the wild-type open reading frame of RSus1 and RSus3 were constructed and transformed into P. pastoris for expression. Both recombinant proteins were purified by DEAE-Sephacel ion-exchange chromatography, Sephacryl S-300 gel filtration chromatography and Resource-Q FPLC system.
The specific activity of recombinant RSuS3 in both sucrose cleavage and synthesis direction were much higher than those of mutated RSuS3 (mRSuS3), and the ratio of sucrose-cleavage to sucrose-synthesis specific activity was also different from mRSuS3. Moreover, the binding affinity for UDPG of RSuS3 was much higher than that of mRSuS3, as revealed by the kinetic analysis data. These results suggested that the mutations, T4P, V39A, D129G and F680S, in mRSuS3 may affect the substrate binding and also the catalytic activity in both sucrose cleavage and synthesis directions.
The kinetic analysis data also showed that the binding affinity of both UDPG and fructose of RSuS1 are much higher than those of mRSuS1. The specific activity of recombinant RSuS1 in sucrose synthesis direction is much higher than that of mRSuS1. The results indicated that the mutations in the C terminus of mRSuS1 may affect the binding of UDPG and fructose to the enzyme and its activity.

目錄 I
縮寫表 IV
摘要 VI
Abstract VII
第一章 研究背景 1
第一節 蔗糖合成酶之生化性質 1
第二節 蔗糖合成酶之生理角色 1
2.1 參與細胞壁多醣類的合成 2
2.2 參與澱粉生合成 2
2.3 參與蔗糖運輸 3
第三節 蔗糖合成酶異構酶 3
第四節 蔗糖合成酶的磷酸化修飾 4
第五節 蔗糖合成酶活性調控因子 5
第六節 蔗糖合成酶基因的調控 5
第七節 水稻蔗糖合成酶的研究 6
第八節 本論文之研究方向及目的 8
第二章 材料與方法 9
第一節 實驗材料與藥品 9
1.1 菌種 9
1.2 質體 9
1.3 藥品 9
第二節 實驗儀器設備 10
2.1 核酸電泳設備 10
2.2 蛋白質電泳與轉印設備 10
2.3 離心機 10
2.4 其他 10
第三節 實驗方法 11
3.1 重組 RSuS 表現質體之建構 11
3.2 質體對大腸桿菌之轉形與轉形株檢定 14
3.3 質體對酵母菌 Pichia pastoris 之轉形與轉形株篩選 15
3.4 重組 RSuS 蛋白質之大量表現 18
3.5 重組 RSuS 蛋白質之純化 18
3.6 重組 RSuS 分析 20
第三章 結果與討論 29
第一節 以酵母菌 Pichia pastoris 表現重組 RSuS1 及 RSuS3 29
1.1 宿主及表現載體 29
1.2 RSuS1 及 RSuS3 表現質體之序列差異 29
1.3 表現載體 pPICZA-RSuS1 及 pPICZA-RSuS3 之建構 30
1.4 P. pastoris 轉形株之鑑定及篩選 30
1.5 P. pastoris 轉形株最適表現條件探討 31
1.6 重組 RSuS1 及 RSuS3 蛋白質之大量表現及純化 32
第二節 重組 RSuS1 及 RSuS3 蛋白質之蔗糖分解與合成方向活性比較 33
第三節 中間代謝物對重組 RSuS1 活性之影響 34
第四節 重組 RSuS1 蛋白質進行 Triton X-114 分層 35
第五節 重組 RSuS1 及 RSuS3 之酵素動力學分析 35
第六節 討論 36
第四章 結論與未來展望 39
第一節 結論 39
第二節 未來展望 39
2.1 野生型 RSuS1 及 RSuS3 生化性質及酵素動力學分析 39
2.2 野生型重組 RSuS1 及 RSuS3 之膜結合性質分析 39
2.3 重組 RSuS3 之 T4P、V39A、D129G 及 F680S 單點突變株之活性分析 40
2.4 RSuS 之結構解析 40
參考文獻 41
圖與表.............................................................................................................................................49

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