Identification And Characterization Of The SG4-R2R3-MYB Genes That Related With Anthocyanin And Lignin Biosynthesis And Regulation In Pear

Fruit peel coloration is an important index of fruit appearance quality,and directly affects the commodity value and market competitiveness of fruit.The accumulation of phenolic compounds in the epidermal cells affects fruit coloration.And anthocyanin accumulation is related with coloration formation of red pear,while lignin accumulation is related with coloration formation of brown pear and formation of pear russeting.Previous studies have shown that biosynthesis of phenols was regulated by R2R3-MYB transcriptional factors.Among,the subgroup four of R2R3 MYB transcriptional factors(subgroup 4 R2R3-MYB,SG4-R2R3-MYB)could regulate the biosynthesis of anthocyanin and lignin negatively.However,related studies were little reported in pear.In this study,through bio-informatics analysis and differential expression analysis,two candidate genes,PbMYB120 and PbMYB140,were identified to be related with biosynthesis of anthocyanin and lignin,respectively.And function verification and regulation mechanism of them were studied.It can provide theoretical basis for pear fruit color control and quality improvement.The main results are shown as follows:1.Identification of the candidate SG4-R2R3-MYB genes that related with regulation of anthocyanin and lignin biosynthesisNine SG4-R2R3-MYB members were identified in pear through selection with the conserved domains.They were clustered into At MYB4-like clade and Fa MYB1-like clade by phylogenetic analysis.At MYB4-like clade includes three members-PbMYB80,PbMYB140 and PbMYB63,which may be involved in the regulation of the lignin pathway and the general phenylpropanoid pathway.Fa MYB1-like clade includes six members-PbMYB19,PbMYB107,PbMYB76,PbMYB49,PbMYB121 and PbMYB120,which may be involved in the regulation of the flavonoid pathway.Predictive analysis of protein interaction networks also found that At MYB4-like clade member,PbMYB80,may be involved in the regulation of lignin and flavonoids,and Fa MYB1-like clade members,PbMYB19,PbMYB76 and PbMYB120,may be involved in the regulation of flavonoids.Finally,through differential expression analysis in green pear ‘Zaosu' and its red peel mutation-‘Red Zaosu',PbMYB120 was identified as a candidate gene that may be involved in the regulation of anthocyanin biosynthesis;through differential expression analysis in brown pear-‘Niitaka' and its green peel mutation-‘Suisho',PbMYB140 was identified as a candidate gene that may be involved in the regulation of lignin biosynthesis.2.PbMYB120 negatively regulate anthocyanin biosynthesis through direct repression and indirect repressionTransient over-expression of PbMYB120 in ‘Red Bartlett' delayed the coloration of‘Red Bartlett',anthocyanin content and Pb UFGT expression were also suppressed in its transient over-expressed fruit.Yeast one hybrid and dual-luciferase assays found that PbMYB120 could bind to the promoter of Pb UFGT,and repress its promoter activity.These results suggested that PbMYB120 could inhibit anthocyanin biosynthesis directly.Yeast two hybrid and bimolecular fluorescence complementation assays found that PbMYB120 could interact with Pbb HLH3 and Pbb HLH33.Dual-luciferase assays found that the addition of PbMYB120,could repress the activation of of Pb UFGT promoter activity by the activator-type MYB-b HLH complexes.These results suggested that PbMYB120 could also inhibit anthocyanin biosynthesis indirectly.3.Mode of PbMYB120 expression is consistent with that of anthocyanin accumulationThe degree of red coloration in pear depends on the anthocyanin content,which is determined by the biosynthesis,transportation,degradation and consumption of anthocyanin.Among them,anthocyanin biosynthesis is regulated by both the positive and negative regulatory factors.The analysis of multiple red-skin pear cultivars found that change of PbMYB120 expression was consistent with that of anthocyanin accumulation.During the early exposure stages of ‘Red Zaosu',light strongly induces the expression of PbMYB10 and PbMYB10 b,which promotes the expression of structural genes in the anthocyanin biosynthesis pathway and thus induce anthocyanin biosynthesis;in the late stage,expressions of PbMYB10 and PbMYB10 b were not changed,while the expression of PbMYB120 continued to increase,this may be a protective mechanism to prevent excessive accumulation of anthocyanin.4.PbMYB140 regulates lignin biosynthesis positivelyLignin is a key factor in the formation of pear russeting.In this study,PbMYB140 was found to be higher expressed in the russet peel than in the non-russet peel,suggests it is involved in the regulation of lignin biosynthesis.Transient over-expression of PbMYB140 in pear fruit induced the expression of many genes in lignin biosynthesis.And stable over-expression of PbMYB140 in pear root induced the deposition of lignin in the vascular tissue.All of the above results showed that PbMYB140 could induce lignin biosynthesis.Dual-luciferase assay showed that PbMYB140 could activate promoter activities of Pb C3 H and Pb CCo AOMT,indicates that PbMYB140 could regulate lignin biosynthesis directly.In addition,promoter activity of PbMYB140 could be activated by PbMYB83.And transient over-expression assay also indicated that PbMYB83 could induce the expression of many genes involved in lignin biosynthesis through activation of PbMYB140 and PbMYB58 gene expression.