Application of no utility plant resources by a recombinant DNA technology

该项目的重点是通过重组DNA技术在根癌作物叶片中积累有价值的蛋白质，这些蛋白质即使在收获时也具有光合作用和代谢活动。目标植物和酶以日本流行的典型根生作物甘薯和植物来源酶辣根过氧化物酶为研究对象。人工基因表达系统的研制是实现这一目标的重要基础技术。1)辣根过氧化物酶基因prxC1a和prxC2在甘薯中的导入和表达，将prxC1a和prxC2基因连接到CaMV35S启动子下游，与潮霉素抗性基因一起导入甘薯愈伤组织中。2)诱导基因表达系统的构建。在SWEA…中积累大量有用的蛋白质在不损害马铃薯基本代谢活性的情况下，需要更多的马铃薯叶片，需要处于适宜发育阶段的转基因诱导系统。其中一种方法是利用拟南芥HSP18.2基因的热诱导启动子。将HSP18.2启动子与GUS报告基因连接，导入烟草培养细胞(Nicotinana Tabacum By2)。当培养温度从27℃筛选到35℃时，GUS活性在2小时后增加了1,000倍，对该体系的改进将有助于我们的目标。其诱导机制已被阐明。温氏链霉菌合成结构性阻遏蛋白，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。通过将VB与抑制子结合来释放抑制。因此，在烟草BY-2细胞中，CaMV35S启动子驱动了阻遏蛋白的结构基因，并检测到阻遏蛋白的积累。将该操纵子序列放置在GUS结构基因上游的不同位置，并将其导入含有该抑制基因的BY2细胞的核染色体上。在BY-2细胞的培养液中加入VB可显著诱导GUS活性。确定了GUS基因上游引入的操作子序列的数目和位置的最佳化。该系统将用于马铃薯块茎发育后叶片喷施VB生产马铃薯过氧化物酶的实际生产。较少

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