不同花色绿绒蒿DFR基因克隆及表达分析

Cloning and Expression Analysis of DFR Gene from Meconopsis with Different Colors

二氢黄酮4-还原酶(DFR)是花青素合成通路中的关键酶,其表达与花青素积累紧密相关,影响花朵呈色。为了探究DFR基因与绿绒蒿花色形成的关系,以红色的红花绿绒蒿和黄色的全缘叶绿绒蒿为试验材料,使用RT-PCR技术成功从中克隆出DFR基因,并对其进行生物信息学及RT-qPCR分析。结果显示,2种绿绒蒿DFR基因的cDNA全长为1 131,1 125 bp,分别编码376,374个氨基酸,命名为MpDFR、MiDFR。生物信息学分析发现,2种蛋白均属于亲水性蛋白,且具有DFR特有的NADPH结合结构域和底物特异性结合结构域。系统进化树表明,MpDFR、MiDFR蛋白与鸦片罂粟、沉水樟、三枝九叶草、澳洲坚果、蒂罗花的亲缘关系最近。Motif分析发现,DFR蛋白基序在不同植物中均较为保守。RT-qPCR结果显示,MpDFR、MiDFR在不同组织中均有表达,具有组织特异性。花蕾期中MpDFR、MiDFR基因表达量最高,且显著高于其他2个时期。另外,红花绿绒蒿中DFR表达量均高于全缘叶绿绒蒿。结合西南林业大学屈燕课题组研究发现,这2种绿绒蒿中花青素类化合物的积累模式与DFR基因的表达模式一致,所以,研究花青素生物合成途径中的关键结构基因DFR对于了解该途径以及通过基因工程手段改良植物花色均十分重要。

Dihydroflavone 4-reductase(DFR)is a key enzyme in the anthocyanidin synthesis pathway.Its expression is closely related to the accumulation of anthocyanidin,which affects flower color.In order to explore the relationship between DFR gene and the formation of flower color in Meconopsis,red-flowered M.punicea and yellow-flowered M.integrifolia were selected as experimental materials.The DFR gene was successfully cloned from them using RT-PCR technology,and bioinformatics and RT-qPCR analysis were performed on them.The results showed that the cDNA full-length of the two DFR genes in Meconopsis was 1131,1125 bp,respectively,encoding 376 and 374 amino acids,named MpDFR and MiDFR.Bioinformatics analysis revealed that both proteins belonged to hydrophilic proteins and possessed the unique NADPH-binding domain and substrate-specific binding domain of DFR.The phylogenetic tree indicated that MpDFR and MiDFR proteins had the closest genetic relationships with Papaver somniferum,Cinnamomum micranthum,Epimedium sagittatum,Macadamia integrifolia and Telopea speciosissima.Motif analysis found that the DFR protein motif is relatively conserved in different plants.The RT-qPCR results showed that MpDFR and MiDFR were expressed in different tissues with tissue specificity.During the bud stage,the expression levels of MpDFR and MiDFR genes were the highest and significantly higher than the other two stages.In addition,the expression level of DFR in M.punicea is higher than that in M.integrifolia.Based on the research findings of Quyan′s research team at Southwest Forestry University,it was found that the accumulation pattern of anthocyanin-like compounds in these two species of Meconopsis was consistent with the expression pattern of the DFR gene.Therefore,studying the function of DFR genes is crucial for gaining a deeper understanding of anthocyanin biosynthesis pathways and improving plant flower color through genetic engineering methods.