The Mechanism Of PbLAC4-like Gene Involve In The Degradation Of Anthocyanin In Pear

There are red color fading phenomenons during the development of pear leaves such as ‘Zaosu’,the ‘Zaosu’ and ‘Red Zaosu’ petals,and the ‘Red Zaosu’ receptacles.The color fading phenomenon is related to the degradation of anthocyanin.So far,there are not many studies on the degradation mechanism of anthocyanin in pear.Previous research in our laboratory has screened out the PbLAC4-like gene that may be related to the degradation of anthocyanin from the transcriptome data of the ‘Red Zaosu’ receptacles in different periods.This gene can encode laccase4-like in pear,which is a polyphenol oxidase containing three copper oxidation domains.On this basis,the function of PbLAC4-like gene in the degradation of anthocyanin was verified in this study,and the upstream regulatory factor was preliminarily screened.The main results are as follows:1.The expression level of PbLAC4-like gene was significantly negatively correlated with the content of anthocyanin and chlorogenic acid during the color fading process of pear leaves,petals and receptacles.In this study,the expression level of PbLAC4-like gene and the contents of anthocyanin,catechin,epicatechin,procyanidin B2 and chlorogenic acid in pear leaves,petals and receptacles with discoloration were analyzed.During the process of red color fading in three different tissues,the expression trend of PbLAC4-like gene was generally increased,while the content of anthocyanin and chlorogenic acid was decreased.The correlation analysis results showed that the decrease of anthocyanin and chlorogenic acid content in pear leaves,petals and receptacles was significantly correlated with PbLAC4-like.In addition,the phylogenetic analysis of PbLAC4-like protein showed that PbLAC4-like protein had high homology with the LAC involved in flavonoid degrading in Litchi and Arabidopsis thaliana.Therefore,next,we studied whether PbLAC4-like was involved in the degradation of anthocyanin and chlorogenic acid.2.PbLAC4-like was directly involved in the degradation of anthocyanin in pear and may indirectly affect the accumulation of chlorogenic acid.The transient overexpression of PbLAC4-like in ’Palacer’ fruitlet peel significantly reduced the content of anthocyanin and chlorogenic acid compared with the control.This experiment verified that PbLAC4-like was significantly correlated with the reduction of anthocyanin and chlorogenic acid content in pear.PbLAC4-like protein was obtained by prokaryotic expression,the protein after prokaryotic expression of empty vector was taken as the control,and the activity of PbLAC4-like protein was measured with anthocyanin and chlorogenic acid standard samples as substrates respectively.The results showed that the activity of PbLAC4-like protein in degrading anthocyanin was about 4 times that of the control,while the activity of degrading chlorogenic acid was not significantly different from that of the control.Therefore,we could learn that PbLAC4-like degraded directly anthocyanin resulting in the color fading of pear leaves,petals,receptacles,and its influence on chlorogenic acid content may be indirect.In addition,we measured the degradation activity of anthocyanin in five pear leaves in different periods,and found that its activity was basically consistent with the expression trend of PbLAC4-like,which further proved that PbLAC4-like plays an important role in the degradation of pear anthocyanin.3.PbMYB26 might be involved in the degradation of anthocyanin in pear by regulating PbLAC4-like.Through the phylogenetic analysis and quantitative detection of candidate upstream regulatory factors,we could learn that only the expression pattern of PbMYB26 was consistent with PbLAC4-like in pear leaves,petals and receptacles.Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 could directly combine the promoter of PbLAC4-like,enhance the promoter activity,so as to improve the expression level of PbLAC4-like.Then,we verified this conclusion in vivo.Overexpression of PbMYB26 in the ’Zaosu’ pear pericarp significantly promoted the expression of PbLAC4-like,and the expression of PbLAC4-like in the pericarp of PbMYB26 overexpression was about 4.5 times that of the control.