Studies on inhibition of apple ripening using antisense technique

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Apple is also a typical climacteric fruit. Therefore, it is appeared to be possible to extend the storage life of apple fruit by inhibiting ethylene biosynthesis using antisense technique. Furthermore, we are interested in molecular mechanism of the differences of the storagelongevity among apple cultivars. Lay-Yee and Knighton (1995) reported afull-length cDNA (MdACS-1) encoding ACS from ripening apple. Recently, two more cDNAs of ACS (MdACS-2, MdACS-3) from ripening apple were isolated (Rosenfield et al., 1997). Thus, it is considered that apple has also a multigene family of ACS to control ripening process like tomato. However, their genomic sequences are unknown yet. As a first step for the elucidation of the mechanism controlling the storage longevity, the sequence of gene encoding the MdACS-1 gene was isolated by screening a genomic library from Malus domestica L.Borkh cv Golden Delicious. A probe DNA was made from PCR using primers designed from MdACS-1 cDNA sequence and 'Golden Delicious' genomic DNA as a template. Three of 13 positive phage clones were appeared to contain the full length of the gene coding regions. One of them (1-6) contained partialy a restriction map which was identical to that of the cDNA.The sequencing of the subcloned fragment (5.6kb) revealed the presence of MdACS-1 gene which consists of exons and three introns. The number and size of four exons and location of introns are similar to other ACS genes isolated from tomato, rice, and Arabidopsis (Lincoln et al., 1993 ; Zarembinski and Theologis, 1993 ; Abel et al., 1995). The sequence also included 2,111b and 1,011b of the 5'-and 3'-flanking regions, respectively. We constructed a chimeric gene containing the full-length promotor region fuged to the coding sequence for the GUS gene. By using this characterization of the promotor is currently in progress.