Development of recycling biomass for recycling systems of organic waste

将6种同工酶中编码强纤溶蛋白水解酶的前体型和活性体同工酶A的基因亚克隆到pEU3-NII中，并在小麦胚性无细胞蛋白质合成系统中进行了表达。原核质粒和活性核质粒分别产生了相对分子质量约为26和25 kDa的额外蛋白质，与氨基酸序列计算的相对分子质量相近。将蚯蚓同工酶A的原型基因和活性基因亚克隆到pBI121载体上，构建成含有CaMV35S启动子的融合表达载体pBI121。将这些基因转入拟南芥cv.并获得了T1代种子。将含有活性形式基因的T1种子培养在含有卡那霉素的MS琼脂平板上，观察到了3种类型的植株：1)正常形态，2)裂叶，3)皱叶。所有类型的转基因植株都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代转基因植株也出现了叶片皱缩，叶片逐渐断裂，导致植株死亡。这些结果表明，蚯蚓同工酶A是在植物中产生和工作的，如果同工酶A基因的表达受到足够的启动子的控制，那么就有可能产生有机废物降解植物，这些启动子可以强烈地限制表达，或者对植物生长影响不大的器官特异性表达。

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.