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

本项目由2个小组进行。第一组对与果实品质改善密切相关的酶进行了纯化和克隆试验。测定了苹果果实中山梨糖醇脱氢酶cDNA的碱基序列，该酶可将蔷薇科果实中易位糖中的山梨糖醇转化为果糖。从番茄果实中分离出ACCsynthase 6个同工酶的cdna，并利用各同工酶的特异性抗体对其表达进行了研究。从甜瓜果实中分离到编码ACCsynthase和acccooxidase的cDNA，确定了它们的碱基序列，并通过果实发育对其表达进行了研究。测定了柑橘中与蔗糖的生物合成和降解有关的蔗糖磷酸合酶、蔗糖合酶和酸性转化酶的cDNA碱基序列。从蚕豆种子和芒豆幼苗中分离到具有成熟和衰老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。纯化了与日本梨果软化有关的α -阿拉伯藜苷酶。第二组尝试建立转基因果树的植株再生体系和基因转移方法。以苹果为研究对象，实现了苹果品种愈伤组织再生和农杆菌基因转移，成功转移了S6PDH嵌合基因。以柿子为研究对象，利用愈伤组织和原生质体进行植株再生，通过农杆菌侵染形成标记基因重组体。在芦花科葡萄和柑橘亚科植物中，通过与农杆菌共培养，获得了胚性愈伤组织，并形成了GUS基因重组体。在日本主要水果作物中收集与果实品质密切相关的基因，建立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.