Development of recycling biomass for recycling systems of organic waste

将6种同工酶中强纤溶蛋白酶A同工酶原型和活性型基因克隆到质粒pEU3-NII中，并在小麦无生殖细胞蛋白合成系统中表达。质粒的前形式和活性形式分别产生了分子量约为26和25 kDa的额外蛋白质，其分子量与氨基酸序列计算的分子质量相似。结果表明，该基因可以在植物中表达。将同工酶A的前型和活性型基因亚克隆到质粒pBI121中，得到了与CaMV35S启动子融合的酶基因质粒。这些基因被转移到拟南芥，cv。得到哥伦比亚号和T1号种子。将含有活性型基因的T1种子培养在含有卡那胺的MS培养基琼脂板上，观察3种植株：1)正常形态、2)断裂叶片、3)皱折叶片。所有类型的转基因植株均表达了额外的同工酶A mRNA，但不能产生T2种子。含有pro-form基因的T1种子转基因植株也出现叶片皱褶，叶片逐渐断裂，导致死亡。这些结果表明，蚯蚓同工酶A是在植物中产生并起作用的，如果能通过适当的启动子控制同工酶A基因的表达，对其表达有较强的限制或对植物生长影响不大的器官特异性表达，则有可能产生有机废物降解植物。

The genes encoding pro-form and active-form of strong fibrinolytic protease, isozme A, in the six isozymes of eartworm, Lumbricus rubellus, were subcloned into a plasmid, pEU3-NII, and expressed in the wheat germ cell-free protein synthesis system. Extra proteins with a molecular mass of about 26 and 25 kDa were produced by pro-form and active-form of plasmids, respectively, which are similar to calculated molecular mass from the amino acid sequences. These results shows that the gene from earthworm could express in plant.The pro-form and active-form genes of isozyme A were subcloned into a plasmid pBI121, resulting the plasmids both of which the enzyme genes fused with CaMV35S promoter. These genes were transferred to Arabidopsis thaliana, cv. Columbia, and T1 seeds were obtained. T1 seeds including active-form gene were cultured on MS medium agar plate containing kanamycine and three types of plants, 1) normal shape, 2) breached leaf, 3) wrinkled leaf, were observed. All types of transgenic plants expressed extra isozyme A mRNA, but could not produce T2 seeds. The transgenic plants of T1 seeds including pro-form gene also showed the wrinkled leaves which is breached gradually, resulting the die. These results suggest that the isozyme A from earthworm is produced and works in plants, and the possibility of producing the organic waste-degrading plants if the expression of the isozyme A gene could be controlled by adequate promoters which can restrict the expression strongly or organ-specific expression affecting little on plant growth.