綠竹中與初級細胞壁生合成相關之纖維素合成酶基因探討

Analysis of the Cellulose Synthase Genes Associated with Primary Cell Wall Synthesis in Bambusa oldhamii

陳志祐(Chih-Yu Chen)

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(2010年)

https://doi.org/10.6342/NTU.2010.02971

摘要

在快速生長的竹筍中，細胞壁多醣類的生合成相當旺盛。本論文藉由篩選cDNA庫及RT-PCR，共選殖出10株纖維素合成酶 (cellulose synthase, CesA) 之cDNA (BoCesA1~BoCesA10)，其中7株具有完整的譯讀序列 (open reading frame)，分別可轉譯出1061∼1080個胺基酸（分子量介於 120.77∼122.24 kDa）。 BoCesA1-BoCesA8這8株cDNA彼此在核苷酸序列及胺基酸序列分別有62%-97% 及 67-98% 的同質性，且其胺基酸序列上皆具有植物CesA 序列的特徵，包括：zinc-binding domain、植物CesA所特有的高度變異區域 (HVRI 及HVRII)、八個預測的貫膜部位，及具有高度保守性的UDP-glucose 鍵結部位 (D及DxD motif) 及催化部位 (D 及QxxRW motif) 之可溶性部位。以演化樹分析發現，BoCesA1-BoCesA8 分別屬於三群 (clades)，其中BoCesA1, BoCesA2, BoCesA3及BoCesA8屬於VI-A次群，BoCesA4, BoCesA5屬於V-A次群，而BoCesA6及BoCesA7則屬於I-A次群。
由Southern分析的結果推測BoCesA2、BoCesA5、BoCesA6、BoCesA7在綠竹的染色體中有2-3個拷貝。以quantitative real-time PCR分析此4種BoCesA基因在不同綠竹組織及於試管中培育的綠竹多芽體中之基因表現圖譜，顯示此4種BoCesA基因普遍在綠竹各組織器官中表現，但受到差異性調控。另外，BoCesA基因的表現會受到α-naphthaleneacetic acid的負調控與thidiazuron差異性的影響。由in-situ RT-PCR分析結果顯示出BoCesA2、BoCesA5、BoCesA6及BoCesA7在未出土的竹筍基部及節間是普遍存在，而在綠竹側枝的節間中則是主要表現在維管束中央部位，其中BoCesA2及BoCesA6 mRNA的分佈具空間特異性。藉由TAIL-PCR選殖得BoCesA2 及BoCesA5之5’端序列，以CpGisland2 及CpGplot 程式分析，可在其上搜尋得可能的 core promoter element (CpG island)。此外，在BoCesA2 之轉譯起始點上游序列中，也存在一些類似於參與荷爾蒙調節、維管束專一性表現及光反應有關的cis elements。
另以GST pull-down、yeast two-hybrid 及免疫共沈澱等實驗探討BoCesA是否會與催化UDP-glucose 生成的酵素間具有交互作用。結果顯示， BoCesA7與蔗糖合成酶 (sucrose synthase, SuS) 間具交互作用，但BoCesA7 與UDP-glucose焦磷解酶 (UDP-glucose pyrophosphorylase, UGPase) 間則否。此外，也發現在積貯組織中SuS 與UGPase具交互作用。目前正以雙螢光互補分析法 (biomolecular fluorescence complementation assay) 進一步驗證BoCesA7、BoSuS及BoUGPase 蛋白質間的交互作用。

關鍵字

纖維素合成酶初級細胞壁 ； 綠竹

並列摘要

Synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. Ten cDNAs (BoCesA1-BoCesA10) encoding cellulose synthase (CesA) were cloned from Bambusa oldhamii by screening a bamboo shoot cDNA library and/or by RT-PCR. Among them, seven clones possessed complete open reading frame encoding polypeptides between 1061 and 1080 amino acid residues with relative molecular masses between 120.77 and 122.24 KDa, respectively. BoCesA1-BoCesA8 shared 62-97% identity at the nucleotide level and 67-98% identity at the amino acid level with each other. The deduced amino acid sequences of BoCesA1-BoCesA5, BoCesA7 and BoCesA8 contained the conserved features of plant CesA, including a zinc-binding domain, two plant-specific HVRs (HVR-I and HVR-II), eight potential transmembrane domains, and a soluble cytosolic domain with highly conserved D, DxD and D residues and a QxxRW motif. Phylogenetic analysis of CesAs from different plants suggested that the eight BoCesAs belonged to three clades: BoCesA1, BoCesA2, BoCesA3 and BoCesA8 belonged to clade VI-A; BoCesA4 and BoCesA5 belonged to clade V-A and BoCesA6 and BoCesA7 belonged to clade I-A. Southern blot analysis revealed that BoCesA2, BoCesA5, BoCesA6 and BoCesA7 had 2-3 copies in bamboo genome. The expression profiles of the four BoCesA genes in the different bamboo tissue and the in-vitro cultured bamboo multiple shoots were analyzed by quantitative real-time PCR. The four genes were widely expressed in various bamboo organs and bamboo multiple shoots, but were differentially regulated. They were down-regulated by α-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6 and BoCesA7 mRNAs were all present throughout the base and internode of etiolated shoots that emerged from pseudorhizomes. Expression of the four genes in the internodes of branch shoots that emerged from the nodes of mature bamboo culms was predominantly detected in the center of vascular bundles, and spatial-specific expression was observed with BoCesA2 and BoCesA6. 5'-flanking sequences of BoCesA2 and BoCesA5 cloned by TAIL-PCR contained a core promoter element, CpG Island, which was predicted with the CpGisland2 or CpGplot programs. Several elements with similarity to cis-elements that have been known to be involved in hormone responses, vascular-specific expression and light response were detected in the sequence upstream the translation initiation site of BoCesA2.
GST pull-down assay, yeast two-hybrid system and co-immunoprecipitation were used to investigate whether BoCesA interacts with sucrose synthase (SuS) and UDP-glucose pyrophosphorylase (UGPase). SuS and UGPase are both involved in catalyzing the formation of UDP-glucose for polysaccharide synthesis. The results revealed that protein-protein interactions occurred between BoCesA7 and SuS but not between BoCesA7 and UGPases. In addition, the interactions between SuS and UGPase, which occurred in sink tissues of bamboo, were also observed. Further analyses of the interactions between BoCesA7, SuS and UGPase by bimolecular fluorescence complementation assay are in progress.

並列關鍵字

cellulose synthase ； primary cell wall ； bambusa oldhamii

參考文獻

邱文彬 (2008) 綠竹蔗糖合成酶異構酶之基因表現與生理功能探討, 博士論文, 國立台灣大學, 微生物生化所.

陳志祐（2003）綠竹筍纖維素合成酶cDNA 之分子檢定, 碩士論文, 國立台灣大學農業化學系

Amor Y, Haigler CH, Johnson S, Wainscott M, Delmer DP. (1995). A membrane-associated form of sucrose synthase and its potential role in synthesis of cellulose and callose in plants. Proceedings of the National Academy of Science USA 92(20): 9353-9357.

Appenzeller L, Doblin M, Barreiro R, Wang H, Niu X, Kollipara K, Carrigan L, Tomes D, Chapman M, Dhugga KS. (2004). Cellulose synthesis in maize: Isolation and expression analysis of the cellulose synthase (CesA) gene family. Cellulose 11: 287-299.

Arioli T, Peng L, Betzner A, Burn J, Wittke W. (1998). Molecular analysis of cellulose biosynthesis in Arabidopsis. Science 279: 717-720.