| Pear is one of the most important commercial fruits,and widely distributed throughout of China.Organic acid is one of the standards for intrinsic quality evaluation of pear.There are a variety of pear cultivars in our country and different cultivars have differences in the accumulation of organic acids.In the past years,a lot of researches have been carried out,focusing on the the components and content of organic acids,however,it was hardly reported about genes involved in the regulation of organic acids accumulation in pear fruit.In the present study,first,we analyzed the difference of organic acid content of mature pear fruits among the nine varieties from five major cultivation system using HPLC,confirming malic acid and citric acid were the two principal organic acids,then PbrALMT9,PbrTDT1 and PbrVHA-c4 were cloned from‘Dangshansuli’(Pyrus bretschneidrei)for further functional anslysis.Main results as following:1.The ripening fruits of nine varieties from five major cultivation system,P.bretschneideri,P.communis,P.pyrifolia,P.ussuriensis and P.sinkiangensis,were collected as samples.The components and content of organic acids in these samples were examined with HPLC method.Results showed that there were at least 7 kinds of organic acids in the pear fruit,including malic acid,citric acid,quinic acid,tartaric acid,oxalic acid,shikimic acid and succinic acid.In all kinds of organic acid,malic acid and citric acid were the predominant organic acids,and the total of their content accounted for 86.68%,which were higher than other kinds of organic acid.Among the five different cultivations,P.communis exhibited the highest content of organic acid,P.ussuriensis and P.sinkiangensis were moderate,while P.bretschneideri and P.pyrifolia were lower.Results of the correlation analysis indicated that there was a more significant difference between the content of malic acid and citric acid and the content of total organic acid.In addition,the content of malic and the content of citric acid was very significantly correlated,as well as the content between shikimic acid and succinic acid.2.We analyzed the Aluminum-activated malate transporter(ALMT)family in six main Rosaceae species and screened out the PbrALMT9 gene,related to the organic acid accumulation in Chinese white pear.In this study,we performed a comprehensive analysis of ALMT genes for five Rosaceae species:Pyrus bretschneideri(pear),Malus domestica(apple),Fragaria vesca(strawberry),Prunus persica(peach)and Prunus mume(mei).A total of 113 genes were identified and named as ALMT according to a similar method in Arabidopsis.All ALMT genes were grouped into 3 large subfamiles and ten small subfamilies based on the analysis of phylogenetic tree and conserved structure.Synteny analysis indicated that the duplication models of most genes in pear and apple were WGD or segmental duplication,implying that WGD was the primary driving force for gene family expansion in pear and apple,while the main duplication pattern was dispersed duplication in strawberry,peach and mei.In addition,All Ka/Ks values were less than 1 among 7 synteny gene pairs in pear illustrated the continuous purifying selection played a key role in keeping the stable function of PbrALMTs generated from duplication event.On the basis of RNA-seq database,combing the changes of malate during the pear fruit development and the expression patterns of PbrALMTs,we found three genes,belonging to the group B,were associated with the malate metabolism,confirming the fact that purifying selection could maintain the stability of the function.Furthermore,PbrALMT9 not only showed the highest expression level among these thee genes,also had a positive change tendency accompanied by the changes of the malate content during the whole development stages.For better comprehend the mechanism of PbrALMT9 involved in regulating the malate accumulation,we performed a verification by transgene technology.The results were consistent with our expectations.Taken together,wide-genome analysis provided an overview for ALMT gene family in six Rosaceae species,and the result of transgene laid a foundation for further functional analysis.3.PbrTDT1 was cloned from‘Dangshansuli’(P.bretschneideri),containing an opening reading frame(ORF)of 1590bp and encoding 530 amino acids with predicted protein molecular weight(Mw)57.59 KDa and isoelectric point(pI)7.22.Motif scanning indicated that PbrTDT1 included a typical Na_sulph_symp domain.Phylogenetic tree analysis revealed that PbrTDT1 had a higher homology with AtTDT.PbrTDT1 localized in the vacuolar and contained 13 transmembrane regions.PbrTDT1 was related to the malate accumulation during the pear fruit ripening.Overexpression of PbrTDT1 in tomato,transgenic lines showed higher malate content compared to the WT.On the contrary,in RNA interference lines,the content of malate was lower than overexpression transgenic lines.Simultaneously,we noticed that PbrTDT1 could also increased the content of fumarate and decreased the content of citrate.4.PbrVHA-c4 was cloned from ’Dangshansuli’(P.bretschneideri),containing an ORF of 501 bp and encoding 166 amino acids with predicted protein Mw 16.76 KDa and pI 8.68.Phylogenetic tree analysis demonstrated that PbrVHA-c4 had a higher homology with AtVHA-c4 and CitVHA-c4.PbrVHA-c4 localized in the vacuolar and contained 4 transmembrane regions.The analysis of expression pattern revealed that PbrVHA-c4 had a close relationship with the accumulation of citrate.Overexpression of PbrVHA-c4 in tomato also verified that.In addition,the results of yeast two hybrid(Y2H)and Bimolecular fluorescence complementation(BiFC)displayed that PbrVHA-c4 had an interaction with PbrERF13,which was an ethylene response transcriptional factor and might be related to improved the citrate concentration. |
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