Identification and Expression Analysis of Sugar Metabolism Genes in Apple

Candidate genes were identified by performing Blastp analysis against apple gene set (nucleic acid), in the Malus Genome Database from ‘Fondazione Edmund Mach Istituto Agrario San Michele All'Adige’, Italy (http://genomics.research.iasma.it/blast/blast.html) [18] (link) using A. thaliana invertase, SUSY, HK, SPS, SUT, TMT and vGT sequences (obtained from The Arabidopsis Information Resource (http://arabidopsis.org/) as query (except for FK using Lycopersicon esculentum[19] as query), and an E-value of 1,00E-04 as threshold. The putative candidate gene sequences were retrieved from the Malus Genome Database: http://genomics.research.iasma.it/gb2/gbrowse/apple/. The corresponding sequences of candidate genes were then used for a BLAST search against the Malus EST database in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) to confirm that each predicted gene is expressed in Malus transcriptome while there is a high similarity EST sequence (score >300 bp, and identity >98%). Then, all ESTs sharing high similarity (>98%) with predicted genes were subjected to contig assembly (score >300 bp, and identity >98%; http://mobyle.pasteur.fr/cgi-bin/MobylePortal/). After similarity analysis between the predicted gene and its EST-constructed contig or EST, the divergent gene in splicing again underwent a Blastp analysis against all predictions in apple (nucleic acid) (http://genomics.research.iasma.it/blast/blast.html) using EST-constructed contig or EST sequence so that a concordant sequence with EST would be found in all predictions. Forty-one putative candidate genes involved in sugar metabolism in apple, including 3 CWINVs, 3 NINVs, 3 vAINVs (Table S1), 5 SUSYs (Table S2), 4 FKs (Table S3), 6 HKs (Table S4), 6 SPSs (Table S5), 5 SUTs, 5 TMTs and 2 vGTs (Table S6), were screened for expression analysis. Additionally, representative MdSOTs (MdSOT1, Genbank accession, AY237401, low Km; MdSOT2, AY237400, high Km [20] ) and MdSDHs were also used for expression analysis. Although 17 predicted SDH homology genes were found in Malus genome [18] (link), only MdSDH1 to MdSDH9 (SDH1, AY244806; SDH2, AY244807; SDH3, AY244809; SDH4, AY053504; SDH5, AY244811; SDH7, AY244813, SDH8; AY244812; SDH9, AY244810) had been systematically investigated as NAD-dependent sorbitol dehydrogenase [21] , [22] (link). Since MdSDH2 shares high similarity of cDNA sequence with MdSDH3 to MdSDH9[18] (link), a pair of universal primers was designed for MdSDH2-SDH9 based on their conserved cDNA region.