Navarro F, Florencio F J
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, Spain.
Plant Physiol. 1996 Aug;111(4):1067-75. doi: 10.1104/pp.111.4.1067.
The gene encoding thioredoxin in the facultative heterotrophic cyanobacterium Synechocytis sp. PCC 6803 (trxA) has been cloned by heterologous hybridization using the corresponding gene trxM from the cyanobacterium Anacystis nidulans as a probe. The deduced amino acid sequence of trxA predicts a protein of relative molecular weight of 11,750 and has strong identity with its cyanobacterial counterparts and other m-type thioredoxins of photo-synthetic eukaryotes. The trxA gene has been expressed Escherichia coli as a functional protein of 12 kD and has been shown by western blot analysis to be the same size as in Synechocystis. The trxA gene is transcribed in Synechocystis as a single transcript of 450 nucleotides and accumulates to the same level under photosynthetic and heterotrophic growth conditions. The trxA gene was inactivated with a kanamycin-resistant cassette, and total segregation of the disrupted trxA gene was obtained only when the trxM gene from A. nidulans (E.D.G. Muller, B.B. Buchanan [1989] J Biol Chem 264: 4008-4014) was simultaneously expressed in Synechocytis. Our results suggest an essential role of thioredoxin not only when cells grow photosynthetically but also under heterotrophic conditions.
利用来自蓝藻集胞藻6803(trxA)的硫氧还蛋白基因,以蓝藻念珠藻的相应基因trxM作为探针,通过异源杂交进行了克隆。trxA推导的氨基酸序列预测其相对分子质量为11,750,与蓝藻中的对应序列以及光合真核生物的其他m型硫氧还蛋白具有高度同源性。trxA基因已在大肠杆菌中表达为12 kD的功能性蛋白,蛋白质印迹分析表明其大小与集胞藻中的相同。trxA基因在集胞藻中作为一个450个核苷酸的单一转录本进行转录,在光合和异养生长条件下积累到相同水平。用卡那霉素抗性盒使trxA基因失活,只有当念珠藻的trxM基因(E.D.G. 穆勒,B.B. 布坎南[1989]《生物化学杂志》264: 4008 - 4014)在集胞藻中同时表达时,才获得了被破坏的trxA基因的完全分离。我们的结果表明,硫氧还蛋白不仅在细胞进行光合作用时,而且在异养条件下都起着重要作用。