Prokaryotic Expression Of Anthocyanin Reductase From Tea Flant(Camellia Sinensis (L.) O. Kuntze)

Catechins, as the main component of tea polyphenols and the most importantsecondary metabolites in tea plant， is also the main chemical component to determine thequality of tea. Study of the function of key genes influencing tea catechins biosynthesis isof great significance for tea breeding and the improvement of tea quality.The study of catechins biosynthesis has been nearly completed, ANR, a key enzymein catechins biosynthesis was directly related to the synthesis of EC and EGC. UsingRT-PCR, prokaryotic expression, HPLC technology, we cloned the two sequences of teaANR in GeneBank, expressed their proteins, detected enzyme activity and identified thereaction products. The optimal conditions such as inducing time, temperature, IPTGconcentration, ampicillin concentration were studied.1.The ORF sequences of CsANR1and CsANR2were cloned by RT-PCR, through theuse of DNAMAN, SignalP3.0, SOPMA, SWISS-MODEL Web site, we compared thesequence of the two genes, predicted the signal peptide sequence, and simulated thesecondary and tertiary structure of proteins. The results showed that the sequencesimilarity of CsANR1and CsANR2amino acids was79%.34amino acids of CsANR1’sn-terminal was signal peptide sequence and44amino acids of CsANR2’s n-terminal wassignal peptide sequence. After the removal of signal peptide, the similarity of the twoamino acid sequences was83%. The number and distributions of secondary structuralelements of CsANR1and CsANR2protein secondary structures were similar and thesimulated tertiary structure was also in great similarity. CsANR1and CsANR2had asimilar function by the speculation of protein structure, but the difference of signal peptiderevealed two proteins with different subcellular localization..2. The recombinant plasmids of pET-ANR and pET-ANR2were constructed andtransformed into expression of host rosetta (DE3). The optimal conditions for inducingIPTG were consided as25℃of induction temperature,1.0mmol/L of IPTGconcentration,200μg/mL of ampicillin concentration and5hours of induction time.3. Fusion protein was purified by affinity resin. The enzyme activity of fusion proteinCsANR1and CsANR2was detected taking CYA as the substrate and NADPH as thecoenzyme, and enzymatic reaction products were analyzed by HPLC. The results showedthat fusion protein CsANR1and CsANR2could catalyze CYA into EC, which proved thatboth of them possessed the enzyme activity of ANR.4. The expression of CsANR1and CsANR2in tea leaves at different development stages were studied by qRT-PCR. The rusults showed that the expressions of CsANR1and CsANR2in buds and the third leaf were relatively high.