The Content Of Proanthocyanidin Monomers And Expression Pattern Of ANR/LAR Genes In The Fresh Leaves Of Huangjin Tea

Proanthocyanidin monomers (flavan-3-ols) are one of the most important phenolicgroups in tea, the content and structure types of which affect tea flavour and contributenutrient functions to human. Huangjin tea is a valuable cultivar indigenous to southwestHunan province, but characterization of proanthocyanidin monomer compounds inHuangjin tea remains unclear, meanwhile studies about proanthocyanidin monomerbiosynthesis in this cultivar are limited. In the present work, we aimed to characterizeproanthocyanidin monomers, as well as clone two flavan-3-ols biosynthesis branchgenes in Huangjin tea (cultivar1and2).Methanolic extractions of Huangjin tea leaves were analyzed by HPLC tocharacterize monomers (-)-catachin (C),(-)-epichatachin (EC),(-)-gallocatachin (GC),(-)-epigallocatachin (EGC), and (-)-epigallocatechin gallate (EGCG). In this study,different season and different development stages of proanthocyanidin monomers inHuangjin tea leaves had been investigated. The results showed that in general, the5monomers were the highest in summer, followed by autumn and spring. The profiles ofeach monomer in different season were different. EGC and EGCG were highest inspring, EGC and C were highest in summer, EC and EGCG were highest in autumn.The profiles of each monomer in developing leaves of each season were basically inagreement. When in spring, summer and autumn, the contents of each monomer wereincreased at the early development stages but decreased at late development stages.When in fall, the profiles of each monomer were similar as in summer. The content andprofiles of each monomer in Huangjin tea (cultivar1and2) are no significantdifferences.Two flavan-3-ols biosynthesis branch genes leucoanthocyandin reductase (CsLAR)and anthocyanidin reductase (CsANR) had been cloned from Huangjin tea leaf byRT-PCR. The results of sequencing showed that the PCR products were composed of1044bp and1029nucleotides including the start and stop codon, which were deducedto encode347and342amino acids. The deduced amino acid sequences were analyzedby some bioinformatic softwares, the properties and3D model of CsLAR and CsANRsuggested that they belonged to leucoanthocyanidin reductase and anthocyanidinreductase, respectively.Real-time quantitative PCR had been employed to investigate transcription patternsof CsLAR and CsANR in Huangjin tea leaves. The results showed that, the transcriptionpatterns of CsLAR and CsANR were similar to the contents of each proanthocyanidinmonomer in spring and autumn. In summer, CsANR transcription pattern was similar tothe profiles of EC and EGC in Huangjin tea cultivar1, but CsLAR transcription patternwas not related to that of C and GC. In cultivar2, CsLAR transcription pattern was similar to the profiles of C and GC, but CsANR was not related to that of EC and EGC.Taken together, expression levels of CsLAR in Huangjin tea leaves were similar to thatof CsANR in spring, expression levels of CsLAR were higher than that of CsANR insummer and autumn.In a word, these data demonstrated that EGC and EGCG were the majorproanthocyanidin monomer in the fresh leaves of Huangji tea. Along with season anddevelopment stages of Huangjin tea leaves, the proflies of total proanthocyanidinmonomers were increased at the early, and decreased gradually. LAR and ANRpathway of flavan-3-ols biosynthesis were co-existed in Huangjin tea.