广佛手UDP-鼠李糖合成酶基因的克隆及功能鉴定

Cloning and characterization of a UDP-rhamnose synthase gene from Citrus medica

目的对广佛手Citrus medica var.sarcodactylis中参与合成UDP-鼠李糖的UDP-鼠李糖合成酶(UDP-rhamnose synthase,RHM)基因进行全长cDNA克隆、功能鉴定及差异表达分析。方法基于广佛手转录组数据,筛选和克隆广佛手鼠李糖合成酶基因(CmRHM1)的全长cDNA,并对其进行生物信息学分析;构建原核表达载体,通过体外酶促反应鉴定CmRHMl的功能;利用实时荧光定量PCR分析广佛手不同器官中CmRHM1基因的表达特性。结果CmRHM1基因的开放阅读框(ORF)长度为2007 bp,编码668个氨基酸。该基因编码蛋白的相对分子质量为75330、理论等电点是6.97,为亲水蛋白,无信号肽。多重序列比对显示CmRHMl在序列的N-端和C-端均含有高度保守的2个结构域(GxxGxxG/A和YxxxK)。体外酶促反应结果表明CmRHMl蛋白具有催化UDP-Glc合成UDP-Rha的功能。实时荧光定量结果显示该基因在广佛手的茎、叶和果实中的表达水平依次为叶>果实>茎。结论CmRHM1基因的功能鉴定为UDP-鼠李糖及鼠李糖糖苷的生物合成奠定了物质基础。

Objective To clone and characterize a UDP-rhamnose synthase gene involved in the biosynthetic mechanism of UDP-rhamnose for the medicinal plant Citrus medica.Methods Based on the transcriptome data,the full-length c DNA of UDP-rhamnose synthase gene(CmRHM1)was screened and cloned,and then the bioinformatics analysis was carried out.The prokaryotic expression vector was constructed and the function of CmRHM1 was identified by enzymatic reaction in vitro.Additionally,Real-time PCR was performed to analyze the expressive characteristics of CmRHM1 gene in different plant organs.Results The ORF length of CmRHM1 was 2007 bp,encoding 668 aa.Bioinformatic analysis of the amino acid sequence showed that the molecular weight of encoded protein was 75330,and theoretical isoelectric point was 6.97.This protein was proved to be a hydrophilic protein without signal peptide sequence.Multiple sequence alignment showed that CmRHM1 contained two highly conserved domains(GxxGxxG/A and YxxxK)at the N-terminal and C-terminal regions.The enzymatic reaction in vitro revealed that CmRHM1 has indeed been shown to have catalytic activities for converting UDP-glucose into UDP-rhamnose.The results of qPCR showed that the ranking of the expressive level of the CmRHM1 in the stem,leaf and fruit was leaf>fruit>stem.Conclusion The functional identification of CmRHM1 gene reported here provides the basic foundation for the biosynthesis of UDP-rhamnose and rhamnosides.