多花黄精果糖激酶和GDP-甘露糖焦磷酸化酶的基因克隆及酶结构性质

Cloning and characterization of fructokinase and GDP-mannose pyrophosphorylase genes in Polygonatum cyrtonema Hua

多花黄精(Polygonatum cyrtonema Hua,PC)果糖激酶(PCFRK)和GDP-甘露糖焦磷酸化酶(PCGMPP)是黄精多糖生物合成中重要的关键酶,为探究它们在黄精多糖生物合成途径中的作用,基于转录组测序数据分析,利用RT-PCR技术对这两种关键酶基因进行克隆,并分析酶蛋白的理化性质及结构特点。PCFRK开放读码框(ORF)为1725 bp,编码574个氨基酸,具有两个磷酸果糖激酶B家族的特征保守基序;PCGMPP的ORF为1086 bp,编码361个氨基酸,具有焦磷酸化酶家族的保守位点和活性位点。系统进化树分析发现在已知数据库中PC FRK与深圳拟兰FRK亲缘关系最近;而PC GMPP与芦笋GMPP亲缘关系最近。基因表达分析说明PCFRK和PCGMPP在根状茎中的基因表达总量明显高于其他组织,这与多花黄精根茎中多糖含量高于其他组织是一致的。研究为PCFRK和PCGMPP两种关键酶功能的进一步研究及黄精多糖生物合成途径的解析奠定基础。

Fructokinase(FRK)and GDP-mannose pyrophosphorylase(GMPP)are key enzymes of polysaccharides biosynthesis pathway in Polygonatumcyrtonema Hua(PC).In this study,the two key enzyme genes were cloned using RT-PCR technology based on transcriptome sequencing data,and the properties and structural characteristics of the two enzymes were analyzed.The ORFs of PCFRK and PCGMPP were 1725 and 1086 bp in length and encoded proteins with 574,361 amino acids,respectivel y.PCFRK has two characteristic conserved motifs of the phosphofructokinase B family;PCGMPP had a conserved site and an active site of the pyrophosphorylase family.The homology comparison showed that PC FRK had the highest similarity to the FRK of Apostasiashenzhenica,while PCGMPP had the highest similarity to the GMPP of Asparagus offcinalis.Gene expression analysis showed that the expression level of PCFRK and PC G MPP in rhizome was significantly higher than that in other tissues,which was consistent with the polysaccharide content in rhizome of PC.This study provided the basis for the functional study of PCFRK and PCGMPP and the further study on plant polysaccharides biosynthesis pathway.