Characterization of gene repertoires at mature stage of citrus fruits through random sequencing and analysis of redundant metallothionein-like genes expressed during fruit development.

We carried out a random sequencing of cDNA library derived from mature citrus fruit (Citrus unshiu Marc.) for identifying the gene repertoires expressed at the mature stage. Among 297 clones analyzed, 195 cDNA clones (65.7%) were putatively identified to previously characterized genes with optimized (OPT) scores of >/=200 through a homology search to DNA database, whereas 102 clones (34.3%) resulted in low OPT scores (<200) and did not show any significant sequence identity with previously published genes. Among them, clones homologous to metallothionein (MT)-like genes appeared 62 times, being mostly redundant, and accounting for about 20.9% of the total 297 clones. To gain a better understanding of the MT-like genes, two types of cDNA clones were isolated. One clone (CitMT36) resembled the type 2 MT gene containing Cys-X-Cys motifs in both N- and C-terminal, but the consensus sequence in the N-terminal domain, Cys-Cys and Cys-X-X-Cys was modified in CitMT36 to X-Cys and Cys-X-X-X, respectively. We suggest that these form a 'novel type 2' group of MT-like clones. The other clone (CitMT45) showed homology to type 3 MT-like genes, which have been found in mostly fruit tissues so far. By Southern blot analysis, both clones showed one or two bands, suggesting that both CitMT36 and CitMT45 are present in single or a few copies in the citrus genome. Transcripts of CitMT36 were evenly detected in all tissues examined, whereas those of CitMT45 were detected primarily in fruit during the developmental phase. Neither of the MT-like genes was induced in leaves by Zn and Cu. Collectively, MT-like genes from citrus would be regulated differentially depending on the fruit developmental stage and organs, indicating a change in their expression under the different physiological and molecular environment of fruit cells.