Lee N G, Stein B, Suzuki H, Verma D P
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Plant J. 1993 Apr;3(4):599-606. doi: 10.1046/j.1365-313x.1993.03040599.x.
A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library. Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean. The VN-35 cDNA was inserted in the antisense orientation downstream of the caMV-35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes. Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot. Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR). The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms. Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA. These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant. The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.
从豇豆根瘤cDNA文库中分离出一个编码根瘤特异性尿酸酶（EC 1.7.3.3.）的结节素-35（N-35）cDNA。豇豆尿酸酶（VN-35）cDNA的序列分析显示与大豆的同源性为90%。将VN-35 cDNA以反义方向插入花椰菜花叶病毒35S启动子下游，利用发根农杆菌在豇豆植株上形成转基因毛状根。用慢生根瘤菌（豇豆）感染后，在野生型茎支持的转基因毛状根上产生根瘤。使用聚合酶链反应（PCR）在单个根上的转基因根瘤中检测到反义VN-35 RNA的表达。与用含有β-葡萄糖醛酸酶（GUS）基因的二元载体转化的毛状根上形成的根瘤相比，表达反义VN-35 RNA的根瘤体积较小，尿酸酶活性较低，并且植株表现出缺氮症状。超微结构分析和用抗大豆N-35抗体进行免疫金标记显示，在表达反义VN-35 RNA的转基因根瘤中过氧化物酶体的生长受到抑制。这些数据表明，脲类生物合成的减少限制了共生还原氮对植物的供应。热带豆科植物的根瘤似乎专门用于氮同化，并且在发育上受到控制以产生和运输脲类。
