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Reasrach on Biosynthetic Pathway of Betalains

甜菜碱生物合成途径的研究

基本信息

批准号：
18370017
负责人：
YOSHIHIRO Ozeki
金额：
$ 10.25万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

cDNAs encoding for the homologous gene of DOPA 4,5 dioxygenase (DOD), which was a key enzyme involved in betalain biosynthesis, were isolated from four o'clocks, Arabidopsis, torenia and carnation. The cDNA was introduced into a expression vector and transformed into Escherichia coli In the crude extract prepared from the E. coli, DOD activity was detected to form betalamic acid, indicating that the enzymatic properties of DOD could be elucidated using this extract in vitro. Because betalains were modified by sugar moieties at the intermediate step of cyclo-DOPA of the synthetic pathway, cDNAs for cyclo-DOPA glucosyltransferase (cDOPA5GT) were isolated from four o'clocks, portulaca and globe amaranth. The amino acid sequences of cDOPA5GT cDNAs derived from these plant species consisted of a separated clade from others, suggesting that cDOPA5GT might have arisen early during the evolutionary divergence and established of the Caryophyllales except for Dianthus. The specific activities of other GTs encoded in cDNAs derived from these species isolated simultaneously were analyzed by heterologous expression in E. coli. The GT encoded in a cDNA derived from globe amaranth showed broad specific activity to phenylpropanoids including p-coumaric acid, caffeic acid, sinapic acid and ferulic acid as sugar acceptors. Using this cDNA together with the cDNA of Arabidopsis sucrose synthase, the efficient synthetic method for phenylpropanoid-glucosides was established. These phenylpropanoid-glucosides purified by high performance liquid chromatography could be used as good acyl-donors for anthocyanin acyltransfarase assays.

从四点钟、拟南芥、蓝猪耳和香石竹中克隆了DOPA 4，5双加氧酶（DOD）同源基因的cDNA。DOD是甜菜色素生物合成的关键酶。将该cDNA导入表达载体，转化大肠杆菌。大肠杆菌中，DOD酶活性可形成β-氨丁酸，表明该提取物可用于体外研究DOD的酶学性质。由于甜菜色素在合成途径的环-DOPA的中间步骤被糖部分修饰，因此从四点钟、马齿苋和地球阿马兰斯苋菜中分离环-DOPA葡糖基转移酶（cDOPA 5GT）的cDNA。这些植物cDOPA 5GT cDNA的氨基酸序列组成了一个独立的进化枝，表明cDOPA 5GT可能在进化分化的早期出现，并建立了除石竹外的石竹目。通过在E.杆菌来源于地球阿马兰斯苋的cDNA中编码的GT对作为糖受体的苯丙素类化合物（包括对香豆酸、咖啡酸、芥子酸和阿魏酸）表现出广泛的比活性。将该cDNA与拟南芥蔗糖合成酶cDNA结合，建立了高效合成苯丙素苷类化合物的方法。通过高效液相色谱法纯化的这些苯丙素类葡萄糖苷可用作花青素酰基转移酶测定的良好酰基供体。