丹参F3'5'H基因克隆及其序列分析

目的:为了验证丹参类黄酮-3′,5′-羟基化酶(Flavonoid 3′,5′-hydroxylase,F3′5′H)基因与丹参花色表型的相关性,本研究克隆并分析了紫花丹参99-3株系和一些白花丹参中的F3′5′H基因。方法:本研究通过提取紫花丹参和白花丹参中的总RNA,再将其反转录得到cDNA,以此cDNA为模板,利用PCR方法扩增获得F3′5′H基因全长序列。再利用生物信息学分析方法,分析了该基因编码蛋白质的理化性状、结构域、系统进化等特点,并预测了该蛋白质的亚细胞定位、跨膜区等;利用实时定量PCR方法检测了该基因的表达特异性;利用毛状根遗传转化方法获得了该基因的过表达阳性毛状根株系。结果:F3′5′H基因全长1551 bp,编码516个氨基酸,相对分子质量为57.4 kDa。该基因在丹参花中高丰度表达。在42份(紫花25份;白花17份)丹参样品中发现一个单核苷酸多态性(Single Nucleotide Polymorphisms,SNP)位点在紫花和白花丹参中呈现与花色相关的稳定变化。系统发育分析表明丹参F3′5′H与美女樱的F3′5′H具有较高序列同源性。获得了过表达F3′5′H的毛状根株系。结论:本研究在丹参中克隆到F3′5′H基因,对其序列进行了分析,为进一步研究丹参F3′5′H基因的功能奠定基础。

Cloning and Expression Analysis of F3'5'H from Salvia Miltiorrhiza

To verify the correlation between the Salvia miltiorrhiza F3''5''H (SmF3''5''H) and the phenotype of S.miltiorrhiza flower.This study cloned and analyzed the F3''5''H genes from S.miltiorrhiza 99-3 with purple flowers and some S.miltiorrhiza plants with white flowers.Methods:The total RNA was extracted from S.miltiorrhiza with purple flowers and some S.miltiorrhiza with white flowers.And it was reversed and transcribed into cDNA.Using this cDNA as a template, the full-length of F3''5''H gene order was obtained by PCR method.Bioinformatics analysis was used to analyze the characteristics of physicochemical properties, domains, system evolution and other characteristics.The subcellular localization and transmembrane region etc.of the protein were also predicted.The gene expression features of the genes were detected by real-time quantitative PCR.The transgenic hairy roots of genes were obtained by hairy root genetic transformation method.Results:The length of F3''5''H was 1551 bp, encoding 516 amino acids, and the relative molecular mass was 57.4 kDa.This gene is highly expressed in the flowers of S.miltiorrhiza.One SNP was found in 42 samples (25 purple flowers; 17 white flowers) of S.miltiorrhiza plants.This locus showed a stable change related to flower color in S.miltiorrhiza with purple and white flowers.Phylogenetic analysis showed that F3''5''H had high sequence homology with the F3''5''H of Glandularia x hybrida.The hairy roots over-expressing F3''5''H were obtained.Conclusion:This study cloned and analyzed the F3''5''H from S.miltiorrhiza.These results lay the foundation for further research on the F3''5'' H function of Salvia miltiorrhiza .