Soitamo A J, Zhou G, Clarke A K, Oquist G, Aro E M, Gustafsson P
Department of Biology, University of Turku, Finland.
Plant Mol Biol. 1994 Oct;26(2):709-21. doi: 10.1007/BF00013756.
The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacIQ repressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 mumol photons m-2 s-1 in the presence of 40 or 80 micrograms/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 micrograms/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacIQ system. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.
单细胞蓝藻聚球藻属（Synechococcus sp.）PCC 7942有三个psbA基因，编码光系统II反应中心蛋白D1的两种不同形式（D1:1和D1:2）。在不同光照条件下，这些psbA基因的表达水平以及D1:1和D1:2的合成受到强烈调控。为了更好地理解这些过程背后的调控机制，我们构建了一株能够过量产生psbA mRNA和D1蛋白的聚球藻属（Synechococcus sp.）PCC 7942菌株。在本研究中，我们描述了在psbAI基因前使用tac杂交启动子并结合lacIQ阻遏系统来过量表达D1:1。在40或80微克/毫升异丙基-β-D-硫代半乳糖苷（IPTG）存在的情况下，将细胞在50微摩尔光子·米⁻²·秒⁻¹的光照下培养12小时，可诱导D1:1的过量产生。用IPTG培养的细胞中psbAI mRNA的量和D1:1蛋白的量分别高出三倍和两倍。更高浓度的IPTG（即150微克/毫升）并未进一步增加psbAI信息或D1:1的产生量。D1:1的过量产生导致合成的D1:2水平下降，使得大多数光系统II反应中心含有D1:1。然而，D1:1的过量产生对色素组成（叶绿素a或藻蓝蛋白/细胞数量）或光合作用的光饱和速率没有影响。这以及D1和D2蛋白的总量不受IPTG影响这一事实表明，膜内光系统II中心的数量保持不变。从这些结果中，我们得出结论，psbAI的表达可以通过使用tac启动子和lacIQ系统来调控。然而，D1:1蛋白在膜中的积累受光系统II中心数量的调控。
