Studies on inhibition of apple ripening using antisense technique

苹果也是典型的跃变型果实。因此，利用反义技术抑制乙烯的生物合成有可能延长苹果果实的贮藏寿命。此外，我们还对苹果品种间贮藏寿命差异的分子机制进行了探讨。Lay-Yee和Knighton(1995)报道了苹果成熟过程中编码ACS的全长cDNA(MdACS-1)。最近，又从成熟的苹果中分离到两个ACS基因(MdACS-2、MdACS-3)(Rosenfield et al.，1997)。因此，我们认为苹果和番茄一样，也有一个控制果实成熟过程的多基因家族。然而，它们的基因组序列尚不清楚。作为阐明苹果贮藏寿命控制机制的第一步，通过对金冠苹果基因组文库的筛选，获得了编码MdACS-1基因的序列。以‘金冠’基因组DNA为模板，根据mdacs-1基因序列设计引物，经聚合酶链式反应(PCR)扩增出探针DNA。13个阳性噬菌体克隆中有3个克隆含有全长的基因编码区。对亚克隆片段(5.6kb)进行测序，发现mdacs-1基因由外显子和3个内含子组成。4个外显子的数目、大小和内含子的位置与从番茄、水稻和拟南芥中分离的其他ACS基因相似(林肯等人，1993；Zarembinski和Theologis，1993；Abel等人，1995)。该序列还包括2,111b和1,011b的5‘和3’侧翼区。我们构建了一个融合到GUS基因编码序列的全长启动子区域的嵌合基因。通过使用这一特征，目前正在对推动者进行描述。

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.