Reasrach on Biosynthetic Pathway of Betalains

DOPA 4,5双加氧酶（DOPA 4,5 dioxygenase， DOD）是甜菜素合成的关键酶，从拟南芥、拟南芥和香石竹中分离到编码DOD同源基因的cdna。将cDNA导入表达载体，转化至大肠杆菌，在大肠杆菌制备的粗提物中检测到DOD的活性，形成β -氨基甲酸，表明该提取物可以在体外阐明DOD的酶学性质。由于β素在环- dopa合成途径的中间步骤被糖修饰，因此从四花、马马苋和苋菜中分离到环- dopa葡萄糖基转移酶（cDOPA5GT）的cdna。来自这些植物的cDOPA5GT cdna的氨基酸序列组成了一个与其他植物分离的分支，表明cDOPA5GT可能在进化分化的早期出现，并建立了除石竹属外的石竹属植物。在大肠杆菌中通过异源表达分析了从这些物种同时分离的cdna中编码的其他GTs的特异性活性。从苋菜中提取的cDNA编码的GT对苯丙酸（包括对香豆酸、咖啡酸、辛酸和阿魏酸）具有广泛的糖受体特异性活性。利用该cDNA与拟南芥蔗糖合酶cDNA，建立了苯基丙氨酸糖苷的高效合成方法。通过高效液相色谱法纯化得到的苯丙类糖苷可作为花青素酰基转移酶测定的良好酰基供体。

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.