| Apple is one of the most important processing and table fruits worldwide. China is the largest country which produces freshy apple fruits and concentrated apple juice in the world. However, we are still facing a emergent requirement for special processing varieties with high acidity. In order to investigate the mechanism of malic aicd accumulation in apple fruits, we conducted some researches on several key genes involved in malic acid metabolism and accumulation. The results are shown as follows.1. Isolation and sequcene analysis of 4 key genes involved in malic acid metabolaism.The complete cDNA sequence of NADP–ME,V-ATPase Subunit A,V-PPase and partial sequence of PEPC were isolated from apple fruit. Motif analysis and alignment of predicted amino acid sequence indicated that those sequences were putative candidates.The full-length cDNA of M–ME contained a 2,048 bp ORF encoding a 590 amino acid. The pI and molecular weight of the protein were 5.9 and 64.98 kD, respectively. Compared with other plant homologs in GenBank, M–ME showed more than 90% identity at amino-acid level, especially up to 95% with castor-oil plant . Five NADP-binding motifs existed in M–ME amino acid sequence.The full-length cDNA of MVA–A contained a 1,872 bp ORF which encoded a 623 aa protein with estimated molecular weight of 68.9 kD. Compared with other plant homologs in GenBank, MVA–A showed more than 90% identity at the amino-acid level. Especially with homologs in fruit trees such as Pyrus communis,Prunus persica,citrus and so on, the identity exceeded 95% and reached 99.2% with Pyrus communis from the same subfamily. The highly conserved sequence GAFGCGKT(252-259 aa) also presented in MVA-A amino acid sequence.The full-length cDNA of M–PPi contained a 2280-bp ORF. The ORF encoded a 759-amino-acid protein with estimated molecular weight of 79.4 kD. Compared with other plant homologs in GenBank, M–PPi showed more than 80% identity in amino acid sequence. Three conserved sequences DVGADLVGKVE(246-256 aa),TEYYTS(415-420 aa)and GDTIGD(715-720 aa)also presented in M–PPi amino acid sequence.A 3084-bp partial cDNA of PEPC was cloned and about 110 bp far to the start codon ATG. The inferred amino acid was highly similar with its homologs in other species.2. The transcription of PEPC was down-regulated gradually with fruit development in high-acid fruit, while reverse results appeared in low-acid fruit. Its transcription was higher in high-acid fruit than in low-acid one from 30 to 90 DAB, whereas no obvious difference from 90 to 150.3. In high-acid fruit, M–ME transcripts decreased gradually with fruit development. However, two peaks appeared at 60 and 120 DAB in low-acid fruit. Its transcription was always higher in low-acid fruit than in high-acid one from 60 to 150 DAB.4. The transcription of MVA–A exhibited different pattern in 2 genotypes, i.e. a gradual increase in high-acid fruit but a gradual decrease in low-acid one. Accordingly, its transcription was high in high-acid fruit from 30 to 60 DAB whereas it was high in low-acid one from 90 to 120.5. The transcription of M–PPi exhibited a decreasing trend with development of high-acid fruit, while in low-acid fruit, it decreased after an increasement. Its transcription in high-acid fruit was higher than that in low-acid one during development.6. The recombinant prokaryotic expression construct pET30a-ME pruoduced a 66 kD fusion protein in E.coli BL21.
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