綠竹筍蔗糖磷酯合成酶 cDNA 之選殖與檢定

Molecular Cloning and Characterization of Sucrose Phosphate Synthase from Shoots of Bamboo（Bambusa oldhamii）

洪加正(Chia-Cheng Hung)

指導教授 ： 李平篤

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.02235

摘要

蔗糖磷酯合成酶（Sucrose phosphate synthase，簡稱 SPS）為高等植物組織中調控蔗糖生合成重要酵素，SPS 主要經由磷酸化與異位調控，調節酵素活性。本論文以綠竹筍（Bambusa oldhamii）為材料，進行 SPS cDNA 之選殖與檢定。利用不同物種的蔗糖磷酯合成酶高保守性胺酸序列，設計引子對進行 cDNA 庫篩選用探針之製備，命名為 BPSPS2。從綠竹筍 cDNA 庫中選殖出蔗糖磷酯合成酶 cDNA 長 3225 bp，推導其胺酸序列長 1074 a.a.（BOSPS），並推定其分子量為 119 kDa，pI 值為 5.96。透過已知全長 SPS 胺酸序列，包括 BOSPS ，經由多重序列比對分析，可分成十個區域進行探討。演化分析可知 SPS 來自三個基因家族，分別以 A、B、C 基因家族表示。而 BOSPS 屬於 A 基因家族的一員，其與A 基因家族之同質性達 73-88%。將 BOSPS 序列接到表現載體（pTrcHisB）並選殖到大腸桿菌中進行表現。將表現後之重組蛋白質經純化及活性分析，尚無法測得 BOSPS 之酵素活性。
由南方點墨法分析可知綠竹筍蔗糖磷酯合成酶有 4 個基因套組數。

關鍵字

, 綠竹筍 , 基因家族 ； 蔗糖磷酯合成酶

並列摘要

Sucrose-phosphate synthase (SPS) , a key enzyme in the regulation of sucrose metabolism, is subjected to the regulation by protein phosphorylation and allosterism. In this study，Bamboo（Bambusa oldhamii）cDNA clone was isolated and characterized. According to the conserved sequences of SPS from various plants, primers was designed to amplify a partitive SPS fragment（BPSPS2）for cloning. A 3225-bp cDNA clone （Bambusa oldhamii） encoding sucrose phosphate synthase was isolate from Bamboo shoot cDNA library and conceptually translate into a protein of 1074 amino acid residues with predicted molecular mass of 119 kDa and pI of 5.96. Twenty five full-length plant SPS protein sequences were extracted from the public domain databases, aligned using CLUSTALW and further optimized by eye. it was obtained specific ten regions of interest are highlighted (I–X). Analysis of SPSs from various plants , including Bambusa oldhamii SPS（BOSPS）, showed a well supported separation of known genes into three families, designated A, B and C. BOSPS belongs to Family A and the deduced amino acid sequences showed 73-88% identity with A family. Heterologous expression in Escherichia coli showed that the cDNA clone （BOSPS） is unable to encoded a functional SPS enzyme.

From the Southern hybridization data , it was predicited that the gene family coding for bamboo sucrose phosphate synthase are consistent with four SPS genes.

並列關鍵字

sucrose phosphate synthase , bamboo , Bambusa oldh

參考文獻

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