摘要
该研究基于灰毡毛忍冬常规品种和湘蕾品种转录组数据,筛选到黄酮生物合成关键限速酶基因CHI和CHS的Unigene序列并克隆全长cDNA序列,通过在线生物信息学分析软件和qRT-PCR分析编码蛋白特性和不同花期、不同部位下CHI、CHS基因的表达模式,HPLC测定木犀草苷含量并结合表达量做相关性分析。结果显示,灰毡毛忍冬2个品种中CHI、CHS基因,分别包含627、1170 bp的ORF区,且CHI蛋白和CHS蛋白均为稳定、带亲水性的非分泌型蛋白。qRT-PCR结果表明2个品种CHI、CHS基因在不同花发育阶段及茎、叶中均有差异表达,且在关键花期表达差异明显,结合HPLC数据,木犀草苷含量与基因相对表达量之间呈现负相关性。推测灰毡毛忍冬中CHI、CHS基因参与调控黄酮化合物的累积,可进一步进行其功能研究。该研究首次克隆了灰毡毛忍冬中CHI、CHS基因并进行表达分析,丰富了灰毡毛忍冬黄酮生物合成研究,为进一步探究灰毡毛忍冬中木犀草苷等黄酮类成分差异的分子机制以及良种选育提供研究基础。
In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI)and chalcone synthase(CHS)in the biosynthesis of flavonoids in Lonicera macranthoides,this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and′Xianglei′of L.macranthoides.Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins,and quantitative reverse-transcription polymerase chain reaction(qRT-PCR)to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages.The content of luteo-loside was determined by high performance liquid chromatography(HPLC)and the correlation with the expression of the two genes was analyzed.The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF),respectively,and the CHI protein and CHS protein were stable,hydrophilic,and non-secretory.qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages,particularly the key stages.Based on HPLC data,luteoloside content was in negative correlation with the relative expression of the genes.Thus,CHI and CHS might regulate the accumulation of flavonoids in L.macranthoides,and the specific functions should be further studied.This study cloned CHI and CHS in L.macranthoides and analyzed their expression for the first time,which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L.macranthoides and variety breeding.
作者
曾娟
龙雨青
李灿
曾梅
杨敏
周新茹
刘湘丹
周日宝
ZENG Juan;LONG Yu-qing;LI Can;ZENG Mei;YANG Min;ZHOU Xin-ru;LIU Xiang-dan;ZHOU Ri-bao(School of Pharmacy,Hunan University of Chinese Medicine,Changsha 410208,China;Key Laboratory of Germplasm Resources and Standardized Planting of Hunan Large-scale Genuine Medicinal Materials,Changsha 410208,China;Key Laboratory of Tradional Chinese Medicine Modermization Research in General Colleges and Uniersities of Hunan Prorince,Changsha 410208,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2022年第9期2419-2429,共11页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81673546,81203007)
湖南省自然科学基金项目(2021JJ30515,2021JJ30497)
财政部和农业农村部国家现代农业产业技术体系项目(CARS-21)
2020年湖南省一流专业建设项目。
关键词
灰毡毛忍冬
查尔酮异构酶
查尔酮合成酶
基因克隆
表达分析
含量测定
Lonicera macranthoides
chalcone isomerase
chalcone synthase
gene cloning
expression analysis
content determination
