Studies on inhibition of apple ripening using antisense technique

反义技术抑制苹果成熟的研究

• 批准号: 07660003
• 负责人: HARADA Takeo
• 金额: $ 1.47万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660003/
• 关键词: Apple; ACC shynthase gene; Ethylene; Fruit storage; Antisense; アニチセンス; 再分化; 組換え植物; 遺伝子導入

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.

苹果也是一种典型的跃变型水果。因此，利用反义技术抑制乙烯生物合成，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研究. Lay-Yee和Boghton（1995）报道了来自成熟苹果的编码ACS的全长cDNA（MdACS-1）。最近，从成熟苹果中分离出另外两种ACS的cDNA（MdACS-2，MdACS-3）（Rosenfield等人，1997年）。因此，可以认为苹果和番茄一样，也有一个ACS多基因家族来控制成熟过程。然而，它们的基因组序列尚不清楚。作为阐明控制贮藏寿命的机制的第一步，通过筛选来自苹果（Malus apartica L.Borkh cv Golden Delicious）的基因组文库来分离编码MdACS-1基因的基因序列。使用由MdACS-1 cDNA序列设计的引物和'Golden Delicious'基因组DNA作为模板，通过PCR制备探针DNA。13个阳性噬菌体克隆中有3个似乎含有全长的基因编码区。其中一个（1-6）含有部分与cDNA相同的限制性内切酶图谱，对亚克隆片段（5.6kb）进行测序，结果表明存在MdACS-1基因，该基因由外显子和三个内含子组成。四个外显子的数量和大小以及内含子的位置与从番茄、水稻和拟南芥中分离的其它ACS基因相似（林肯等人，1993 ; Zarembinski and Theologis，1993 ; Abel et al.，1995年）。该序列还分别包括2，111 b和1，011 b的5 '和3'侧翼区。我们构建了一个嵌合基因，含有全长启动子区融合到GUS基因的编码序列。通过使用这种启动子的表征目前正在进行中。

项目成果

Chiba T. et al.: "Transcription of tRNA genes from a large-scale plastid DNA deletion celary reveals the action of nuclear-encoded RNA polymerase in the plastid." J. Plant Physiol.148. 652-656 (1996)

Chiba T. 等人：“大规模质体 DNA 缺失芹菜中 tRNA 基因的转录揭示了质体中核编码 RNA 聚合酶的作用。”