綠竹蔗糖合成酶異構酶之基因表現與生理功能探討

Studies on the gene expression and physiological functions of sucrose synthase isozymes in green bamboo Bambusa oldhamii

邱文彬(Wen-Bin Chiu)

指導教授 ： 王愛玉

國立臺灣大學/生命科學院/微生物與生化學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.10504

摘要

竹子生長十分快速，先前以綠竹 Bamboo oldhamii 的研究顯示蔗糖合成酶SuS 可催化蔗糖與 UDP 反應生成果糖與 UDPG 之可逆反應，在快速生長的竹筍與幼竹中，對於旺盛的細胞壁多醣類合成相當重要，本論文的目的即在探討 SuS 在竹快速生長所扮演的角色。
針對由綠竹筍 cDNA 所選殖出的四株BoSus cDNA (BoSus1、BoSus2、BoSus3 與 BoSus4) 進行序列分析與基因體拷貝數分析，並以 northern 分析、半定量 RT-PCR、免疫組織定位及 in situ RT-PCR 探討各基因在不同生長時期竹筍及不同組織中的表現，此外，也以大腸桿菌表現 4 種重組 BoSuS，並以鈷金屬離子結合膠體 (colbat-based immobilized metal affinity chromatography) 純化，探討不同 BoSuS 之生化特性。
BoSus1 與 BoSus3 在序列上有極高的同質性，兩者可能是重複基因 (duplicate gene) 或是異源同型基因 (homeologous)，同樣的 BoSus2與 BoSus4 序列上也有極高的同質性。動力學上的分析顯示，兩個相似度極高的 BoSuS 異構酶對蔗糖有近似的 Km，但對 UDP 的 Km 則不同。Northern 分析及半定量RT-PCR 結果顯示這四個 BoSus 基因在綠竹各組織部位均有表現，但表現量各自不同，並可發現 BoSus 表現量與竹筍生長的速度成正相關。醣類分析的結果顯示不同生長時期的竹筍各部位之蔗糖濃度的變化趨勢與 BoSus mRNA 及 BoSuS 的表現量變化趨勢呈正相關。
利用水稻多株與單株抗體進行組織定位的研究，在未出土竹筍與葉鞘中之薄壁細胞與維管束、成熟葉中之表皮細胞、葉肉細胞與維管束以及在未展開的葉片 (嫩葉) 的各組織中均可偵測到 BoSuS。以 in situ RT-PCR 探討 4 個基因在未出土竹筍、葉片以及葉鞘中的表現，發現 4 種 BoSus 基因在未出土竹筍與葉鞘的薄壁細胞中均有表現，其中 BoSus2 基因在中間與尖端部位各組織有較明顯的表現差異。在嫩葉與成熟葉各組織中可見 4 種 BoSus 基因表現的差異性，但在葉鞘各組織中 4 種 BoSus 基因則有相近似的表現。
這樣的結果意味著 4 種 BoSuS 在來源與積貯組織均扮演著蔗糖裝載/卸載的角色。BoSuS 在快速生長的綠竹中扮演降解蔗糖以提供細胞壁多醣類合成、澱粉合成所需基質與生長所需能量的角色，而每一種 BoSuS 在不同組織中各有其不同重要性。

關鍵字

綠竹 ； 蔗糖合成酶cDNA 選殖 ； 差異性表現 ； 重組蛋白質 ； 免疫化學染色 ； 組織內RT-PCR

並列摘要

Bamboo is distinguished by its rapid growth. Research on the green bamboo Bambusa oldhamii has suggested that sucrose synthase (SuS), which catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose, is important in providing substrates for the highly active synthesis of cell wall polysaccharides in growing shoots and young culm. The objective of this study is to elucidate the role of SuS in bamboo growth.
Four SuS cDNA clones (BoSus1, BoSus2, BoSus3 and BoSus4) isolated from a cDNA library of etiolated bamboo shoots were characterized by sequences analysis and Southern analysis. Northern analysis, semi-quantitative RT-PCR, immunohistochemical analysis and in situ RT-PCR were used for investigating the expression patterns of each gene in bamboo shoots at various growth stages and in various tissues. In addition, recombinant BoSuS proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography for functional identification.
BoSus1 and BoSus3 may be duplicate or homeologous genes, the sequences of which show high identity. Similarly, BoSus2 shows high identity with BoSus4. Kinetic analysis showed that the two BoSuS isoforms of each type had similar Michaelis constant (Km) values for sucrose, but different values for UDP. Northern analysis and semi-quantitative RT-PCR showed that the four genes were expressed in various bamboo organs but were differentially regulated.
Immunohistochemical analysis using polyclonal and monoclonal antibodies against rice SuS revealed that BoSuS was widely distributed in different tissues of unexpanded leaf. In etiolated shoot and leaf sheath, BoSuS was detected in the parenchyma cells and vascular tissues. In mature leaves, SuS was located in epidermal cells, mesophyll and vascular tissues. In situ RT-PCR analysis showed that the four BoSuS were all expressed in parenchyma cells of leaf sheath and all part of etiolated shoot. The BoSus2 has significant differential expression patterns in the middle and top regions of etiolated shoots. In sink leaf (unexpanded leaf) and mature leaf, the four BoSus gene were also differentially expressed but exhibited similar expression pattern in leaf sheath.
The results underscore the roles of SuS in sucrose loading/unloading in both source and sink organs. Multiple BoSuS isoforms play a variety of important roles in green bamboo, directing translocated carbon towards both the polysaccharide biosynthesis and energy production necessary to support the rapid growth of bamboo.

並列關鍵字

Bambusa oldhamii (bamboo ； cDNA cloning ； Differential gene expression ； Recombinant proteins ； Sucrose synthase ； Immunohistochemistry ； In-situ RT-PCR

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