Application of no utility plant resources by a recombinant DNA technology

本项目重点研究利用重组DNA技术在块根作物叶片中积累有价值的蛋白质，这些蛋白质在收获时也具有光合和代谢活动。目标植物和酶为甘薯、日本常见的典型块根作物巴塔塔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。结果如下：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或prxC2基因cDNA连接到CaMV35S启动子下游，通过农杆菌将prxC1a或prxC2基因与抗潮霉素基因一起导入甘薯愈伤组织。几种抗潮霉素转基因植物的过氧化物酶mRNA、蛋白和酶活性均显著增加。2)诱导型基因表达体系的构建。为了在不损害马铃薯基本代谢活性的前提下，在马铃薯叶片中积累大量有用蛋白质，需要在适宜的发育阶段建立转基因诱导体系。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启动子连接到GUS报告基因上，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1000倍。这个系统的修改将有助于我们的目标。维吉尼亚霉素的生物合成是由维吉尼亚丁醇内酯（VB）诱导的。诱导的机理已被阐明。vinginiae合成组成抑制蛋白，该抑制蛋白结合到维吉尼亚霉素生物合成酶编码区上游的操作序列上。抑制通过VB与抑制因子结合而释放。因此，在烟草BY2细胞中，CaMV35s启动子驱动了该抑制蛋白的结构基因，并检测了该抑制蛋白的积累。该操作序列被放置在GUS结构基因上游的不同位置，并引入到含有抑制基因的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。建立了GUS基因上游引入算子序列数目和位置的优化方案。该系统可用于马铃薯块茎发育后叶片上喷施VB生产过氧化物酶。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