Analysis On The Function Of PbrAHA6 And PbrAHA8 In Regulation Organic Acids Accumulation In Pear

After synthesis,most of the organic acids in fruit are transported to vacuoles by transporter proteins,ion channels or proton pumps.The study of proton pumps related to the transport of organic acids plays an important role in understanding the molecular regulation mechanism of organic acid accumulation.Based on the published genome-wide sequences of seven Rosaceae species,the information of P-type ATPase gene family was identified and analyzed by bioinformatics method;In addition,we explored the relationship between the P3A-ATPases(PbrAHAs)expression and organic acid content,and identified the members of P3A-ATPases who play an important role in the accumulation of organic acid in pear fruit.In order to verified the function,we made the subcellular localization,transient transformation and tomato heterologous genetic transformation.The results as follows:1.419 P-type ATPase genes were identified in seven Rosaceae species(Chinese white pear,apple,peach,strawberry,Japanese apricot,European pear and Du pear),and divided into five subfamilies according to phylogenetic analysis: P1 B,P2B,P2 A,P3A,P4 and P5.Different subfamilies have different conserved motifs and cis-acting elements,which may lead to functional divergence within different subfamilies.The results of collinearity analysis showed that there were 441 pairs of collinearity genes in P-type ATPase gene family in seven Rosaceae species.Dispersed duplication and Whole-genome duplication were the main driving forces of P-type ATPase family gene expansion in pear(Chinese white pear,European pear and Du Pear)and apple,while dispersed duplication and transposed duplication played a major role in P-type ATPase family gene expansion in strawberry,peach and Japanese apricot.Purification selection is the main driving force of gene evolution of P-type ATPase family.In summary,the bioinformatics analysis of Ptype ATPase gene family lays a solid foundation for understanding the evolution and functions of P-type ATPase genes in Rosaceae.2.Through qRT-PCR analysis of the expression of P3A-ATPases(PbrAHAs)in the fruit development of ‘Dangshansuli’,8 of 14 PbrAHAs were expressed in the fruit development,but the expression level of 6 genes were almost 0.The contents of citric acid and malic acid in ‘Dangshansuli’ of the same period were determined by high performance liquid chromatography(HPLC),the correlation analysis showed that there was a significant positive correlation between PbrAHA8 and citric acid content during fruit development,and PbrAHA6 was positively correlated with the malic acid content during the development of pear,suggesting PbrAHA8 and PbrAHA6 are play an important role in the accumulation of citric acid and malic acid in fruit during fruit development.3.In order to further study the function of PbrAHA6 and PbrAHA8 on the process of organic acid accumulation,this study constructed the 35s-PbrAHAs-GFP expression vector and transformed to tobacco leaves,subcellular location shows that PbrAHA6 and PbrAHA8 are localized on the vacuolar membrane,the results suggested that PbrAHA6 and PbrAHA8 might be participated in the accumulation of organic acid in the vacuole;Overexpression of PbrAHA6 and PbrAHA8 transient transformation to pear fruits can cause a significant increase of citric acid and malic acid content,and inhibition of PbrAHA6 and PbrAHA8 gene transient transformation to pear fruits cause a significant decrease of citric acid and malic acid content.Constructed p BI121-PbrAHA6 and p BI121-PbrAHA8 plant overexpression vector,the PbrAHA6 and PbrAHA8 transformation of tomato which in the method of agrobacterium tumefaciens infection,the contents of malic acid and citric acid in transgenic tomato fruits were significantly increased by compare to WT.Through dual luciferase assay demonstrated that five transcription factors were identified that could regulate the expression of PbrAHA8 and one transcription factor were identified that could regulate the expression of PbrAHA6,which lays an important foundation for further research on the function of PbrAHA8 and PbrAHA6.