Application of no utility plant resources by a recombinant DNA technology

该项目的重点是通过重组DNA技术在根作物叶片中积累有价值的蛋白质，即使在收获时也具有光合和代谢活性。目标植物和酶是甘薯、日本流行的典型块根作物甘薯和有用的植物源酶辣根过氧化物酶。人工基因表达系统的开发是实现这一目标的重要基础技术。1）将辣根过氧化物酶基因prxC 1a和prxC 2的cDNA分别连接到CaMV 35 S启动子的下游，通过农杆菌介导法将其与潮霉素抗性基因一起导入甘薯愈伤组织中。2）诱导型基因表达系统的构建 关于我们 要使马铃薯叶片基本代谢活性不受损害，需要在适宜的发育时期建立转基因诱导体系。方法之一是利用拟南芥HSP 18.2基因的热诱导启动子。将HSP 18.2启动子与GUS报告基因连接，并导入烟草培养细胞（Nicotinana tabacum BY2）中。当培养温度从27℃升至35℃时，2 h后GUS活性提高了1,000倍，对该体系的改进将有助于我们的目标。诱导机制已经阐明。酿酒酵母合成组成型阻遏蛋白，阻遏蛋白与维吉尼亚霉素生物合成酶编码区上游的操纵子序列结合。通过VB与阻遏物的结合来释放阻遏。因此，在烟草BY2细胞中，CaMV 35 s启动子驱动阻遏物的结构基因，并检测到阻遏物蛋白的积累。将操纵子序列置于GUS结构基因上游的不同位置，并导入到含有阻遏基因的BY2细胞的核染色体上。在BY2细胞培养基中添加VB可显著诱导GUS活性。确定了操纵子序列的数目和导入GUS基因上游的位置的最佳化。该系统可用于马铃薯块茎发育后叶面喷施VB生产甘薯过氧化物酶。少

The project focus on the accumulation of valuable proteins in leaves of rootcrop, which have photosynthetic and metabolic activities even at the harvesting, by a recombinant DNA technology. Target plant and enzyme were sweat potato, Ipomoea batatas as a typical rootcrop popular in Japan and horseradish peroxidase as a useful plant origin enzyme. Development of artificial gene expression system is an important basic technology in this purpose. The following results were obtained :1) Introduction and expression of horseradish peroxidase genes, prxC1a andprxC2 in sweat potato.The cDNA of prxC1a or prxC2 gene was ligated downstream of CaMV35S promoter and introduced to callus of sweat potato together with hygromycin-resistant gene using Agrobacterium. Several transgenic plants resistance to hygromycin showed significant accumulation of mRNA, protein and enzyme activity of the peroxidase.2) Construction of inducible gene expression system.To accumulate high amount of useful proteins in swea … More t potato leaves without damage of basic metabolic activity, inducible system of transgenes at suitable developing stage is required. One of approach is application of heat-inducible promoter of Arabidopsis thaliana HSP18.2 gene. The HSP18.2 promoter was ligated to GUS reporter gene and introduced to tobacco cultured cells (Nicotinana tabacum BY2). When incubation temperature was sifted from 27℃to 35℃, GUS activity increased 1,000 times after 2 hr.Modification of this system will be useful for our goal.Biosynthesis of virginiamycin is induced by vinginia butanolides (VB) in Streptomyces virginiae. The mechanism of induction was elucidated already. S.vinginiae synthesizes constitutively repressor protein and the repressor binds to operator sequence upstream a coding region of virginiamycin biosynthetic enzymes. Repression is released by binding of VB to the repressor. Therefore, a structure gene of the repressor was driven by CaMV35s promoter in tobacco BY2 cells, and accumulation of the repressor protein was detected. The operator sequence was placed in various positions upstream GUS structure gene, and introduced to nudear chromosome of BY2 cells harboring the repressor gene. Addition of VB in culture medium of the BY2 cells induced GUS activity significantly. Optinization of number of the operator sequence and position to be introduced upstream of the GUS gene was established. The system will be used to practical production of peroxidase in sweat potato by spraying of VB on the leaves after development of potato tuber. Less