比利时杜鹃类黄酮3-O-糖基转移酶基因的克隆、原核表达及功能分析

Cloning, prokaryotic expression, and functional validation of flavonoid 3-O-glycosyltransferase gene (Rh3GT) from Rhododendron hybridum Hort

摘要:

类黄酮3-O-糖基转移酶是植物花青素糖苷化的关键酶。为研究杜鹃花3GT基因的功能，本研究从红比利时杜鹃中克隆了一个3GT基因开放阅读框(open reading frame,ORF)命名为Rh3GT，分析、测定并验证了该ORF及其编码蛋白的理化性质、表达水平、酶学功能等生物学特性。结果表明，Rh3GT全长993 bp，编码330个氨基酸；编码蛋白为亲水稳定的弱酸性蛋白，隶属糖基转移酶家族(GT-B型)，PSPG box结构域中第44位为谷氨酰胺(Q)；系统进化分析发现比利时杜鹃Rh3GT与越橘Vc3GT和黑果越橘Vm3GT的亲缘关系最近；Rh3GT在根中几乎不表达，在茎、叶、花中均有表达，且在盛开期花瓣中表达量最高；花瓣瞬时过表达Rh3GT后发现其总花青苷积累也显著增加；Rh3GT重组蛋白在大肠杆菌BL21中成功表达，并以包涵体形式存在，大小约为36 kDa，与理论预测值接近；高效液相色谱检测发现，经过变性纯化和稀释复性后的Rh3GT重组蛋白可催化矢车菊素和UDP-葡萄糖合成矢车菊素3-O-葡萄糖苷，表明表达的蛋白具有3GT活性。本研究为深入探讨杜鹃花花青苷生物合成分子调控机制提供了基础数据，并为潜在的杜鹃花分子育种提供了理论支持。

Abstract:

Flavonoid 3-O-glucosyltransferase (3GT) is a key enzyme in the glucosidation of anthocyanins. To investigate the 3GT gene in rhododendron, we cloned an open reading frame (ORF) of 3GT gene (named Rh3GT) from Rhododendron hybridum Hort (Red cultivar) and then characterized this gene and the deduced protein in terms of the biochemical characteristics, expression level, and enzymatic function. The results showed that Rh3GT had a full length of 993 bp and encoded 330 amino acid residues. The deduced protein was hydrophilic, stable, weak acid, belonging to the glycosyltransferase family (GT-B type), with glutamine (Q) at position 44 in the PSPG box. The phylogenetic analysis showed that Rh3GT was most closely related to Vc3GT from Vaccinium corymbosum and Vm3GT from Vaccinium myrtillus. Rh3GT was expressed in the stems, leaves, and flowers and almost not expressed in the roots, with the highest expression level in petals during full blooming stage. Introduction of pCAMBIAL1302-Rh3GT into petals significantly up-regulated the expression level of Rh3GT and increased the total anthocyanin accumulation. Rh3GT was successfully expressed in Escherichia coli BL21 in the form of inclusion bodies with a size of about 36 kDa. The results of HPLC showed that the recombinant Rh3GT after denaturation, purification, and dilution could catalyze the synthesis of cyanidin and UDP-glucose to synthesize cyanidin 3-O-glucoside, indicating that the expressed protein had 3GT activity. This study provides basic data for further studying the molecular regulation mechanism of anthocyanin biosynthesis and theoretical support for molecular breeding of rhododendron.