Mock H P, Trainotti L, Kruse E, Grimm B
Institute of plant genetics and crop plant research, Gatersleben, Germany.
Plant Mol Biol. 1995 May;28(2):245-56. doi: 10.1007/BF00020244.
We have cloned and sequenced a full-length cDNA for uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) and a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tobacco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and processed to ca. 39 kDa. A truncated cDNA, from which the putative transit peptide had been deleted, was used to over-express the mature UROD in Escherichia coli. Purified protein showed UROD activity, thus providing an adequate source for subsequent enzymatic characterization and inhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination. Maximum UROD-RNA levels were detected in the basal segments relative to the top of the leaf.
我们已克隆并测序了烟草（Nicotiana tabacum L.）尿卟啉原脱羧酶（UROD，EC 4.1.1.37）的全长cDNA以及大麦（Hordeum vulgare L.）的部分cDNA克隆。烟草的cDNA编码一种43 kDa的蛋白质，它与从其他生物体中确定的UROD序列总体相似度为33%。我们推测烟草UROD有一个39个氨基酸残基的N端延伸。这个延伸很可能是一个叶绿体转运序列。UROD的体外翻译产物被导入豌豆叶绿体并加工成约39 kDa。一个缺失了推定转运肽的截短cDNA被用于在大肠杆菌中过表达成熟的UROD。纯化的蛋白质显示出UROD活性，从而为后续的酶学特性研究和抑制研究提供了充足的来源。通过Northern和Western印迹分析，研究了黄化大麦幼苗绿化过程中以及在昼夜循环下生长的大麦初生叶切段中UROD的表达。光照期间RNA和蛋白质的量增加。相对于叶尖，在基部切段中检测到最高的UROD-RNA水平。
