Isolation and transfer of genes of key enzymes to improve the quality of fruit

这个项目是由两个小组进行的。第一组尝试了一些与果实品质改良密切相关的酶的纯化和克隆。测定了苹果果实中山梨醇脱氢酶基因的碱基序列，该酶能将蔷薇科果实中转运糖的山梨醇转化为果糖。在番茄果实中，分离了6种ACC合酶同工酶的cDNA，并用每种同工酶的特异性抗体研究了其表达。从甜瓜果实中分离出编码ACCsynthase和ACCoxidase的基因，测定其碱基序列，并研究其在果实发育过程中的基因表达。测定了编码蔗糖磷酸合成酶、蔗糖合成酶和酸性转化酶的基因的碱基序列，这些基因融合了柑橘转运糖的蔗糖生物合成和降解。从四季豆种子和豌豆幼苗中分离到具有成熟和感官功能的半胱氨酸蛋白酶编码基因，并对其启动子结构和基因表达进行了研究。提纯了与日本梨果实软化有关的α-阿拉伯呋喃糖苷酶。第二组建立了转基因果树的植株再生体系和基因转移方法。在苹果上，实现了苹果品种愈伤组织的植株再生和农杆菌转基因，并成功地转移了S6PDH嵌合基因。在柿上，愈伤组织和原生质体再生植株，通过农杆菌侵染形成标记基因重组。此外，在十字花科葡萄和柑橘亚科中，我们成功地获得了胚性愈伤组织，并通过与农杆菌共培养形成了GUS基因的重组。如上所述，我们可以收集一些与果实品质密切相关的基因，建立日本主要果树的植株再生方法和基因转移方法。

This project was conducted by 2 groups. The first group tried the purification and cloning of some enzymes which concern closely the improvement of fruit quality. The base sequence coding cDNA of sorbitol dehydrogenase which converts sorbitol of translocating sugar in Rosaceae fruit to fructose was determined in apple fruit. In tomato fruit, cDNAs of 6 isozymes of ACCsynthase were isolated and the expressions were investigated using the specific antibody for each isozyme. From melon fruit, the cDNA encoding ACCsynthase and ACCoxidase were isolated, the base sequences were determined and the gene expression were investigated with fruit development. The base sequence of cDNA encoding sucrose phosphate synthase, sucrose synthase and acid invertase concverning the biosynthesis and degradation of sucrose of translocating sugar in Citrus were determined. The gene encoding cysteine protease which functions the maturation and senscence was isolated from French bean seed and Vigna mungo seedling, and the structure of promotor and the gene expression were investigated. alpha-Arabinofurano sidase concerning the softening of Japanese pear fruit was purified.The second group tried to establish the plant-regeneration system and gene transfer method to for the transgenic fruit tree. In apples, the plant-regeneration from the callus of apple cultivar and the gene transfer via Agrobacterium became possible, and we succeeded the transfer of S6PDH chimeric gene. In persimmon, the plant was regenerated from callus and protoplast, and the recombinant of marker gene was formed through Agrobacterium infection. Further, in grape and citurs belonging sub-family of Rutaceae, we succeeded to get the embryogenic callus and to form the recombinant of GUS gene by co-culturing the callus with Agrobacterium. As described above, we could collect some genes concerning closely the fruit quality, and establish the plant-regeneration method and the gene trans fer method in main fruits crops in Japan.