摘要
【目的】克隆分析三角梅(Bougainvillea spectabilis)二氢黄酮醇-4-还原酶(Dihydroflavonol-4-reductase,DFR)基因(BsDFR),探讨其在三角梅苞片呈色中的作用。【方法】基于三角梅转录组数据,利用PCR技术克隆BsDFR基因,并通过生物信息学工具分析其分子特性;通过分子对接技术预测BsDFR底物特异性;采用实时荧光定量PCR分析该基因在不同颜色三角梅中的表达量差异。【结果】三角梅BsDFR基因(GenBank ID:ON417750)编码区全长987 bp,编码328个氨基酸。BsDFR理论相对分子质量为36.48 kDa,等电点pI为6.33;具有DFR特有的NADPH及底物特异结合位点,属于Asn型DFR;不具有跨膜结构及信号肽,定位于细胞质中;二级结构中α螺旋占比最多,三级结构预测显示为二聚体蛋白。底物对接模拟预测BsDFR对二氢山柰酚(Dihydrokaempferol,DHK)、二氢槲皮素(Dihydroquercetin, DHQ)和二氢杨梅素(Dihydromyricetin, DHM)3种底物均具有催化活性,与结构分析相吻合。进化树分析其与石竹目(Centrospermae)植物聚为一类。qRT-PCR分析发现其在橙色系三角梅中含量较高,进一步推测其主要底物为DHK,催化生成橙色系花青素(天竺葵素)的前体物质——无色天竺葵素苷元。【结论】BsDFR基因是一个典型的植物二氢黄酮醇-4-还原酶基因,主要与橙色系三角梅苞片色素合成有关。
【Objective】The dihydroflavonol-4-reductase(DFR)gene in bracts of Bougainvillea spectabilis was cloned and characterized to study the role it plays in color formation.【Method】BsDFR was cloned based on the transcriptome data on the ornamental plant to study the related bioinformatics.Molecular docking technology was employed to predict the substrate specificity,and qRT-PCR applied to examine the relative transcription levels of the genes in B.spectabilis of different colors.【Result】The full-length coding sequence of BsDFR(GenBank ID:ON417750)was 987 bp encoding 328 amino acids.The protein had a calculated molecular weight of 36.49 kDa and an isoelectric point of 6.33.It had the NADPH and substrate specific binding sites unique to DFR of Asn type without a transmembrane structure or signal peptide.The subcellular localization of the protein indicated it to be cytoplasmic.Alpha helices were the most abundant secondary structure of the protein,while the tertiary structure was a dimer.A substrate docking simulation,consistent with the structural analysis,predicted BsDFR to possess a catalytic activity on dihydrokaempferol,dihydroquercetin,and dihydromyricetin.The phylogenetic tree analysis grouped it along with caryophyllales plants.High expression of the gene was found in the orange B.spectabilis by qRT-PCR.It was speculated that the main substrate to be DHK,which was catalyzed by BsDFR into leucopelargonidin,a precursor of orange-colored anthocyanidin——pelargonidin.【Conclusion】BsDFR in B.spectabilis had typical molecular characteristics of the plant dihydroflavonol-4-reductase,which is associated with the pigment synthesis in the bracts of orange B.spectabilis.
作者
孙蓉
刘桃
潘凯越
刘姗
刁毅
曾道萍
SUN Rong;LIU Tao;PAN Kaiyue;LIU Shan;DIAO Yi;ZENG Daoping(College of Biological and Chemical Engineering,Panzhihua University,Panzhihua,Sichuan 617000,China)
出处
《福建农业学报》
CAS
CSCD
北大核心
2024年第1期33-39,共7页
Fujian Journal of Agricultural Sciences
基金
四川省自然科学基金面上项目(2023NSFSC0145)
大学生创新创业训练计划项目(S202311360061)。
关键词
三角梅
BsDFR基因
生物信息学
表达量分析
Bougainvillea spectabilis Willd
dihydroflavonol-4-reductase gene
bioinformatics
expression analysis
