Isolation And Functional Characterization Of Cytochrome P450 Reductase Genes From Tea Plant

There were many types of cytochrome P450 enzymes in plants,which were widely involved in plant secondary metabolism and plant resistance to adversity stress,and cytochrome P450 enzymes often rely on cytochrome P450 reductase to transfer electrons to exert their activity.Catechins were an important secondary metabolite in tea plants,and their biosynthetic pathway involved multiple cytochrome P450 enzymes,such as CsC4H,CsF3’H and CsF3’5’H.The functions of these cytochrome P450 enzymes（CsC4H,CsF3’H and CsF3’5’H）had been proven,but the cytochrome P450 reductases（CPRs）that support the activity of these enzymes in tea plants have not yet been reported.Their supporting effect on the cytochrome P450 enzymes in the synthetic pathway of tea plant catechins was not clear.To this end,this study first isolated 3 gene sequences encoding cytochrome P450reductase（CsCPR1,CsCPR2 and CsCPR3）from the tea plant genome by homologous sequence alignment,and used the fresh leaf c DNA of’Shaancha 1’.These genes were cloned as templates,and bioinformatics analysis and subcellular localization were performed on them;then they were expressed in prokaryotic cells to analyze their electron transfer activities;and then they were co-expressed with CsC4H and CsF3’H in yeast to verify their function in vivo;Finally analyzed the correlation of their expression in different leaf tissues of tea plants and different degrees of shading treatment with the accumulation of catechins,the results were as follows:1.The open reading frame of CsCPR1 is 2079 bp,encoding 692 amino acids,the molecular weight was 76.85 k Da,and the isoelectric point was 5.15;the open reading frame of CsCPR2 was 2112 bp in length,encoding 703 amino acids,the molecular weight was 78.28 k Da,and the isoelectric point was 5.23;The open reading frame of CsCPR3 was2130 bp in length,encoding 709 amino acids,with a molecular weight of 78.48 k Da and an isoelectric point of 5.36.The alignment of homologous sequences showed that three CsCPRs contained cytochrome P450 reductase conserved domains,namely membrane anchoring regions,P450 enzyme binding regions,FAD,FMN and NADPH binding regions.Phylogenetic tree analysis showed that CsCPR1 belongs to CPRⅠfamily,and CsCPR2and CsCPR3 belong to CPRⅡfamily.2.The rCsCPRs recombinant protein was obtained by prokaryotic expression.In vitro enzymatic reactions showed that rCsCPR1,rCsCPR2 and rCsCPR3 could all use NADPH as the electron donor,and transfer electrons to oxidized cytochrome c to generate reduced cytochrome c,indicating that all three CsCPRs have electron transfer activity.The K_mvalues of rCsCPR1,rCsCPR2,and rCsCPR3 to cytochrome c were 124.12,74.93 and 49.68μM,respectively,and the V_maxvalues were 5.55,3.76 and 2.93μM?min^-1,respectively.The k_cat values were 420.43,285.31 and 221.96 min^-1,respectively.The efficiencies(k_cat/K_m)were 3.39,3.84 and 4.47μM^-1?min^-1,respectively,and rCsCPR3 had the highest catalytic efficiency for cytochrome c.3.Through eukaryotic expression,CsCPRs and CsC4H orCsF3’H are co-transformed into yeast strain INVSC1,after inducing protein expression,the substrate was fed,and the product was detected by HPLC.All three CsCPRs can support CsC4H activity.The substrate cinnamic acid was transformed into a product to coumaric acid.CsCPR1 has the strongest supporting activity forCsC4H.The co-expression strain of CsCPR1 and CsC4H can convert 52.45% of cinnamic acid into coumarin acid.The three CsCPRs can also support the CsF3’H activity.The substrate naringenin was transformed into the product eriodictyol.CsCPR2 has the strongest support activity forCsF3’H.The co-expression strain of CsCPR2 and CsF3’H can reduce 86.68%of the activity.Naringenin was converted into eriodictyol.This shows that the three CsCPRs can help cytochrome P450 enzymes to transfer electrons to exert their activity,but different cytochrome P450 enzymes require different CsCPRs to transfer electrons to exert their maximum activity.4.The results of qRT-PCR analysis showed that CsCPRs were expressed in different leaf tissues,and the content was higher in mature leaf tissues.After 30%and 50%shading treatments,the expression of CsCPR1 did not change much,and both CsCPR2 and CsCPR3 showed an increase trend.Their expression pattern were significantly related to the accumulation pattern of catechins,which may be involved in the biosynthesis of catechins in tea plants.