The Function Of PbPH5 And PbPH3 Gene Involve In The Malic Acid Accumulation In Pear Fruits

Flavor of pear（Pyrus）fruit is affected by many factors,one of the most important factors is organic acid,and different kinds of organic acids are related with various taste.The synthesis mechanism of organic acid in pear fruit has been reported,but regulation mechanism of accumulation remains unclear.In this study,functional gene and transcription factor involved in malic acid accumulation in pear fruit were selected from different pear germplasm resources.This study will helpful to analyze the molecular regulation mechanism of pear fruit acidity and lay the foundation for quality control and genetic improvement during pear fruit development.The main results are as follows:1.PbPH5 participates in the accumulation of malic acid in pear fruit.In recent years,the function of P-type proton pump gene has been gradually analyzed.The P3 subfamily has two branches,and P_3Aparticipates in the transmembrane transport of H⁺.Using the subfamily of P_3A-type ATPase genes of Arabidopsis as the query for a BLAST search,we identified all of the pear P_3A-ATPase genes.Then the content of organic acids and relative expression level during different development stages of various pear cultivars were measured,which were‘Dangshansuli’,‘Zaosu’,‘Whangkeumbae’,‘Abate Fetel’and‘Jinxiangshui’.The related gene PbPH5 was screened by correlation analysis,which was significantly positively correlated with the change trend of malic acid content.And the correlation coefficients in various pear cultivars were 0.915**,0.932**,0.900**,0.656*,and 0.518*（P<0.05 or P<0.01）,respectively.It was found that PbPH5-GFP protein was located in the vacuolar membrane by overexpressed the PbPH5-GFP gene in protoplasts of Arabidopsis.On the basis of the phylogenetic relationships,this gene was highly homologous with other reported homologous genes,such as Petunia hybrida,Malus domestica,and Citrus reticulata etc.,indicating that the function of this gene was highly conserved.The content of malic acid at the injection site was significantly increased when overexpression of PbPH5 in the mature fruit of‘Zaosu’.On the contrast,RNA interference of PbPH5 in pear significantly decreased the PbPH5 transcript level and the malic acid content as well.Subsequently,the function of PbPH5 was further verified by stable genetic transformation of tomato（Lycopersicon esculentum）.The results showed that the content of organic acids in mature fruit of tomato were significantly increased when overexpression of this gene,compared with the control.The results showed that PbPH5 may participate in the accumulation of organic acids in pear fruit.2.PbPH3 involved in the accumulation of organic acids in pear fruits.Through the correlation analysis,five candidate genes that may be involved in the regulation of fruit acidity were screened in‘Dangshansuli’.The result of phylogenetic analysis showed that Pb WRKY26（a）,which named PbPH3,had the closest relationship with other transcription factors,and correlation analysis in more cultivars,‘Hongsucui’,‘Cuiguan’,‘Qiuxiang’and‘Hanhong’,showed that the expression level of PbPH3 was significantly correlated with the change trend of malic acid content.And the correlation coefficients in various pear cultivars were 0.871**,0.748*,0.889**and 0.910**（P<0.05 or P<0.01）,respectively.In pear callus of stable overexpression PbPH3 gene,malic acid content was increased significantly,but the expression of PbPH5 did not change significantly.The results of dual luciferase assay and yeast single hybridization further verified that PbPH3 could not directly bind to the promoter of PbPH5.Real-time quantitative PCR was used to detect the gene expression of ALMT and MDH families in transgenic callus.The results showed that some genes in these two families were up-regulated or down-regulated,indicating that PbPH3 may participate in the accumulation of organic acids in pear fruit through other molecular pathways.