Reasrach on Biosynthetic Pathway of Betalains

从拟南芥、香石竹和香石竹中分离到DOPA-4，5-双加氧酶(DOD)的同源基因，DOD是甜菜碱生物合成的关键酶。将该基因导入表达载体，转化到大肠杆菌中，检测DOD活性形成倍他氨基酸，表明该粗提物可用于体外研究DOD的酶学性质。由于甜菜碱在合成途径的环-多巴中间步骤中被糖基修饰，因此从马齿苋和籽实中分离到了环-多巴葡萄糖基转移酶(CDOPA5GT)的cDNA。来自这些植物的cDOPA5GT的氨基酸序列由一个独立的分支组成，表明cDOPA5GT可能在石竹目植物进化分化的早期出现，并在石竹目植物中建立。通过在大肠杆菌中的异源表达，分析了从这些同时分离的物种中获得的cDNA中编码的其他GT的比活性。从苋菜中提取的cDNA中编码的GT对苯丙素类化合物表现出广泛的特异性活性，包括作为糖受体的对香豆酸、咖啡酸、芥子酸和阿魏酸。将该基因与拟南芥蔗糖合成酶基因结合，建立了苯丙素类糖苷的高效合成方法。经高效液相色谱分离纯化的苯丙烷基葡萄糖苷可作为花青素酰基转移酶测定的良好的酰基供体。

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.