Reynolds P H, Smith L A, Dickson J M, Jones W T, Jones S D, Rodber K A, Carne A, Liddane C P
Department of Scientific and Industrial Research-Fruit and Trees, Palmerston North, New Zealand.
Plant Mol Biol. 1992 Jun;19(3):465-72. doi: 10.1007/BF00023394.
Two isoenzymic forms of aspartate aminotransferase are present in the plant fraction of developing lupin root nodules. One of these forms, aspartate aminotransferase-P2 (AAT-P2), increases dramatically with the onset of biological nitrogen fixation and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. A day 18 lupin nodule cDNA library in the lambda ZapII vector was immunoscreened with a monoclonal antibody specific for AAT-P2 and yielded two near-full-length 1700 bp clones. These clones were sequenced. Amino acid sequences from three peptides derived from immunopurified AAT-P2 were aligned, and showed 100% homology with the amino acid sequence deduced from the cDNA clones. The DNA sequence showed 50% homology with AAT sequences from a range of animal sources. Conversion of the clones to the phagemid form allowed their expression in Escherichia coli where both exhibited enzyme activity that could be immunoprecipitated with AAT-P2-specific monoclonal antibodies. Western blot analysis revealed protein moieties with molecular masses of 39, 43, 45 and 55 kDa. The 5' end of the clones coded for a hydrophobic leader sequence of about 50 amino acids indicative of a targeting sequence and consistent with the plastid localisation of nodule AAT-P2.
在发育中的羽扇豆根瘤的植物部分存在两种天冬氨酸转氨酶同工酶形式。其中一种形式，即天冬氨酸转氨酶 - P2（AAT - P2），随着生物固氮作用的开始而显著增加，并且与豆科植物 - 根瘤菌共生体系中植物对氨的同化作用相关。用对AAT - P2特异的单克隆抗体对λZapII载体中的18日龄羽扇豆根瘤cDNA文库进行免疫筛选，得到了两个近全长的1700 bp克隆。对这些克隆进行了测序。将从免疫纯化的AAT - P2衍生的三个肽段的氨基酸序列进行比对，结果显示与从cDNA克隆推导的氨基酸序列具有100%的同源性。该DNA序列与一系列动物来源的AAT序列具有50%的同源性。将克隆转化为噬菌粒形式使其能够在大肠杆菌中表达，二者均表现出可用AAT - P2特异单克隆抗体进行免疫沉淀的酶活性。蛋白质印迹分析揭示了分子量为39、43、45和55 kDa的蛋白质部分。克隆的5'端编码一个约50个氨基酸的疏水前导序列，这表明是一个靶向序列，并且与根瘤AAT - P2的质体定位一致。
