| Acidity is an important part of fruit quality,affecting the taste of apples and other fruits.Apples are one of the most important fruit trees in temperate regions,the difference in acidity of different apple germplasm resources fruits is obvious.Screening and functional analysis of candidate genes for fruits acidity in apple germplasm resources are of great significance for improving fruit quality.In this paper,the content of organic acids in 101 apple genus was determined using high performance liquid chromatography varieties with significant differences in acidity traits(high acid and low acid)has been selected.In these varieties,transcriptome sequencing was performed on the fruits of Malus toringoides which has special organic acid changes at different developmental stages.The PEPP(Pyrophosphate-energised proton pump),a candidate gene for fruits acidity,was screened out.And explored the gene function,the main results obtained are as follows:(1)The distribution of organic acid content in wild and cultivated apple fruits was determined,and it was found that the acid content of wild apple fruits was significantly higher than cultivated apple.Wild apples contain both malic acid and citric acid,the content of malic acid is 2.58 to 29.27 mg/g FW,and the content of citric acid is 0 to 24.21 mg/g FW.The content of malic acid in cultivated apple fruit is 1.72 to 10.10 mg/g FW.Discovered the germplasm Malus toringoides with a special trend of citric acid and malic acid during fruit development.With the development of the fruit,the malic acid content of Malus toringoides fruit showed a downward trend,but the citric acid content increased significantly.(2)Transcriptome analysis was performed on fruits of Malus toringoides at different development stages,and three databases with Q30 values greater than 88% were obtained.The three databases were compared respectively,and 3337 differential genes coexisting in the three comparative groups were identified,and the differential genes in each comparative group are about 14000.Among these differential genes,the gene MDP0000280551 which is more consistent with the accumulation pattern of total organic acid content was selected.This gene encodes a pyrophosphatase proton pump,named PEPP.(3)The mRNA sequence of the PEPP gene in apple is 4845 bp,including 7 introns and 8 exons,and the c DNA length is 2304 bp.The coding region of MtPEPP was isolated from the c DNA library of Malus toringoides fruit,and the matching degree of sequencing was 97%.Subcellular localization results of Arabidopsis protoplasts showed that MtPEPP was localized on the mitochondrial membrane(4)MtPEPP was constructed on the p GWB 411 vector with strong 35 S promoter,and a positive strain of MtPEPP heterologously expressing ‘Micro-Tom' tomato was obtained.The citric acid content of mature fruit in transgenic lines 13 and 21 was 2.2 mg/g FW higher than wild tomato(6.5 mg/g FW),and total acidity was also 2.6% higher than wild tomato(10.4%).The plant height of tomato plants heterologously expressing MtPEPP is lower than that of wild tomatoes,and the fruit plumpness is reduced.(5)The coding region of the MtPEPP gene was connected to the p MDC83 vector with double 35 S promoter,and the positive pulp callus overexpressing MtPEPP in the apple pulp callus of Wanglin was obtained by Agrobacterium transformation.It was found that the highest malic acid content of the overexpressed apple pulp callus is twice higher than the wild type,and the highest total acidity could reach 10.6%,while the wild type had only 7.5%. |
PDF Full Text Request