Transcirption Factors Analysis In Flavonoid Biosynthesis Pathway And Gene Function Of ANR In Tea Plant Camellia Sinensis (L.) O. Kuntze]

Phenolic compounds, an important secondary metabolite in tea [Camellia sinensis（L.）O. Kuntze], account for18%to36%dry weight in the fresh leaves and tender stemsand could be differentiated into several subgroups including phenolic acid, flavonols,flavan-3-ol (Catechin), flavones, anthocyanins, proanthocyanidins (PAs) and otherspecialized forms of flavonoids. Most phenolic compounds share similar biosyntheticpathways such as shikimic acid pathway, phenylpropanoid pathway and flavonoidsynthetic pathway, in which R2R3-MYB, bHLH, and WD40proteins have been shown tocontrol multiple enzymatic steps in these biosynthetic pathways. However, few relatedtranscription factor genes have been documented in Camellia sinensis. Meanwhile, thefunction of anthocyanidin reductase (ANR) gene is still uncertain. This paper focuses onbioinformatical analyzing transcription factor genes in tea genome, followed by figuringout the gene function of ANR by using tobacco genetic transformation system.The main research contents were as follows:1. The presence of R2R3-MYB, bHLH, and WD40were statistically andbioinformatically analyzed on127,094unigenes in tea genome.Identify each unigene by its conserved domain, resulting in identification ofR2R3-MYB (73), bHLH (49), and WD40(134), respectively.A phylogenetic tree was constructed for R2R3-MYB proteins from Arabidopsis andtea genome. R2R3-MYBs were further divided into27subgroups (Sg), Sg26and Sg27were expanded compared to Arabidopsis data. Motifs in some R2R3-MYB subgroups ofwere redefined according to MEME.Six genes in Sg4might be representative factors for phenolic acid metabolism andlignin biosynthesis. Two genes in Sg5might regulate PA biosynthesis. One gene in Sg7may play its roles in flavonol biosynthesis regulation.32subfamilies were divided on basis of the phylogenetic tree constructing for bHLHproteins from Arabidopsis and tea genome. bHLH proteins from tea genome were groupedinto9subfamilies. According to the functional annotation of Arabidopsis, two bHLH genesin subfamily2and five genes in subfamily24were predicted to involving in flavonoidbiosynthesis pathway.Analysis of CsMYB4-1, CsMYB4-2, CsMYB4-3, CsMYB4-4, CsMYB5-1, andCsMYB5-2revealed the interaction motif [DE]Lx2[RK]x3Lx6Lx3R, potentiallycontributing to the specificity of the bHLH partner in the stable MYB–bHLH complex. The wide evolutionary gap prevented phylogenetic analysis of WD40s; however, asingle gene, CsWD40-1, was observed to share80.4%sequence homogeny with AtTTG1.2. Full length validation and qRT-PCR analysisFull length end-to-end PCR and qRT-PCR were used to validate selected genes andgenerate relative expression ratio profiles in tea leaves by developmental stage andtreatment conditions, including hormones（GA, ABA） and wound treatments. Potentialtarget binding sites were predicted.3. Identification of ANR genes in tea genome, bioinformatics analysis, qRT-PCRanalysis and promoters cloningTwo ANR genes in tea genome have been identified, the sequences of which wereconsistent with GU944768and JN024667in NCBI, and renamed by CsANR1and CsANR2in this paper. Evolutionary tree showed that CsANR1and CsANR2were closely related toVvANR and DkANR. Protein structure analysis of CsANR1and CsANR2showed thatboth of them have the highest consistency with crystal structure of grape ANR3hfsB.NADPH binding sites, substrate-binding sites and catalytic sites of CsANR1and CsANR2were predicted according to the bioinformatics analysis. Promoters of CsANR1andCsANR2were cloned, the length were2464bp and1500bp, respectively. Cis-actingelements responsible to light, hormones and environmental stress were found in eachpromoter. CsANR1and CsANR2were highly expressed in buds, the fourth leaves and theroots; CsANR1and CsANR2were found obvious response to light and sugar; theexpression of CsANR1and CsANR2response to hormones and wound were not obvious.4. Function of ANR genes analysis by transgenic tobacco.The flowers of genetically modified tobacco lines become shallow with varyingdegrees, in which colors of transgenic tobacco lines of CsANR1were much lighter.Meanwhile, the contents of anthocyanin in genetically modified tobacco flowers weredeclined and the contents of PAs were increased compared to the wild flowers. qRT-PCRwas used to validate the expression of relative genes involving in flavonoid biosynthesispathway. In transgentic flowers of CsANR1, the expressions of CHS, CHI, F3’H weredown-regulated, which F3’H the most significant. The expression of ANS wasremarkablely up-regulated; In transgentic flowers of CsANR2, the expressions of CHS,CHI, F3H, ANS, DFR were down-regulated. However, the expression of F3’H wassignificantly up-regulated.5. Function analysis of the most probably regulatory protein CsMYB5-2targeting onANR gene. Bioinformatics analysis showed that CsMYB5-2was closely related to DkMYB4(BAI49721) and PtMYB115(EEE81917). Transgenic tobacco lines of CsMYB5-2wereobtained by transforming through Agrobacterium. The contents of PAs were significantlyaccumulated in the flowers of trans-CsMYB5-2tobacco. The highly up-regulatedexpression of ANR in the flowers of trans-CsMYB5-2tobacco hinted the regulatoryfunction of transcription factor CsMYB5-2working on ANR gene.