| Phloridzin is the characteristic phenolic substance and is a phytoalexin that provides resistance to plant pathogens such as Venturia inaequalis(Cke.) Wint. and Erwinia amylovora. Phloridzin has been recognized as an antioxidant, a potential anti-diabetes agent. In addition, phloridzin has been widely used in human medicine relating to obesity, cardiovascular disease. UDP-glucose: phloretin-2’-O-Glycosyltransferase from Malus x domestica(MdP2’GT) catalyzes the transfer of phloretin to phloridzin, which is a key enzyme in phloridzin biosynthesis. It may be helpful to indicate how to regulate the synthesis of phloridzin and provide a theoretical basis for improving the quality of apple through regulating the level of phloridzin by the means of cultivation and genetics.Using a PCR-based cloning strategy,three MdP2’GT genes were cloned and their bioinformatics were analyzed. The difference of MdP2’GT genes expression between apple cultivars was studied, and the relationship between the gene expression and the synthesis of phloridzin was discussed. The main research results were listed as follows.1. It was shown that the cDNA clones for UGT71A15, UGT71K1 and UGT88F1 were respectively 1416,1434 and 1452 nucletides in length; there were more α-helix, accounting for 40-46%,and there were typically Rossmann folds in amino terminal and carboxy terminal, containing PLN02992 domain; MdP2’GTs were hydrophilic, existed in chloroplast or cytoplasm and had no transmembrane domain or signal peptide. Amino acid sequence of UGT88F1 shared the high homology with UGT88 F subfamily(79.9-99%), but low homology with UGT88 subfamily(34.2-53%). Amino acid sequence of UGT71A15 shared the high homology with UGT71A16(92%), but low homology with UGT subfamily(32.7-58%). Amino acid sequence of UGT71K1 shared the high homology with UGT71K2 subfamily(94%), but low homology with UGT subfamily(33.2-46.6%). The coding region of the isolated UGT88F1 cDNA was subcloned into the pET-32 a vector, resulting in the construction of pET-32a-UGT88F1, then heterologously expressed in E. coli BL21(DE3) induced by IPTG and identified by SDS-PAGE and Western Blot.2. Real-time quantitative PCR analysis indicated that MdP2’GT genes were differentially expressed in bud, flower, leaf, fruit. MdP2’GT genes were detected to be abundant in leaf, bud, apple peel and weak in flower. The expression levels of UGT71A15 and UGT71K1 were higher in leaf, while UGT71K1 and UGT88F1 expression level was higher in apple peel. The expression levels of the three MdP2’GTs reached the maximum at different time and thereafter decreased with age, increased dramatically in senescent leaf. The expression level of UGT71A15 in the peel maintained in low levels and changed in a small scale during fruit development. The expression level of UGT71K1 was detected in high level in young fruit peel, and then decreased to a low level and unchanged until fruit maturity. The expression level of UGT88F1 increased rapidly, thereafter declined dramatically with fruit maturity. The expression of MdP2’GT in leaves during the development were regulated at transcription level, while the expression of MdP2’GT in peel during the development were regulated at transcription and translation level.3. The difference of phloridzin content, MdP2’GT activity was highly cultivar dependent. Phloridzin content was the highest in the leaf, followed by bud, flower, fruit seed, and peel in descenting order. Phloridzin content fluctuated during leaf development. Phloridzin contents in the seeds of the early-maturity cultivars reached the maximum at 55 DAFB, while those of middle-maturity and late-maturity cultivars reached the maximum at 75 DAFB. Phloridzin content and MdP2’GT activity in the fruit peel exhibited a decline as fruit developed. Phloridzin content was positively correlated with MdP2’GT activity in the bud, leaf and fruit peel.4. MdP2’GT was purified 71.0-fold using the ammonium sulfate precipitation, DEAE-Sepharose Fast Flow anion-exchange and Superdex G75 gel filtration chromatography procedures to apparent homogeneity. Specific activity for purified enzyme was 632.0 U/mg and recovery rate was 42.6 %. The molecular weight was estimated to be 50 kDa by SDS-PAGE. The purified enzyme catalyzed the glycosylation reaction under alkaline condition with optimal activity at pH8.5 and was stable over a range of pH 7.0-9.0. Its optimal temperature for enzyme activity was 45 ℃. The Km value for MdP2’GT was determined to be 3.16μM,and Vmax was 0.77 nM/min·mg protein when UDPG(200μM) was used as a sugar donor. At the concentration of 5 mM, MdP2’GT was found to be activated by the presence of Ca2+ and Mg2+ ions. The activity of MdP2’GT was not apparently affected by Na+ or K+, but it was significantly inhibited by Al3+, Cu2+, Mn2+ and Zn2+. The inhibition effect of Cu2+ was the strongest.5. The total phenolic content of the leaves were 26.74-148.99 mg GAE/g DW determinated by Folin-ciocalteu method. It was shown that Qinguan exhibited the best antioxidant properties, whereas Qinyang and Gale Gala possessed better antioxidant properties among seven apple cultivars. The radical scavenging activity of phloridzin was between VC and BHT. The strongest antibacterial activity of apple leaf polyphenols was recorded against Escherichia coli, Staphyloccocus aureus, Salmonella typhimurium with Minimum Inhibitory Concentration(MIC) value of 25 μg/mL, and the lower activity was observed against Shigella and Enterobacter Sakazakii with MIC value of 50 μg/mL. Antibacterial activity of apple leaf polyphenols was remarkably better than any monophenol contained in apples, and 3-4 times better than tea polyphenols. Phloridzin containing no o-dihydroxy structure exhibited lower activity against five tested bacterial strains than other phenols containing o-dihydroxy structure. |
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