Bhalerao R P, Lind L K, Gustafsson P
Department of Plant Physiology, University of Umeå, Sweden.
Plant Mol Biol. 1994 Oct;26(1):313-26. doi: 10.1007/BF00039542.
Two open reading frames denoted as cpcE and cpcF were cloned and sequenced from Synechococcus sp. PCC 6301. The cpcE and cpcF genes are located downstream of the cpcB2A2 gene cluster in the phycobilisome rod operon and can be transcribed independently of the upstream cpcB2A2 gene cluster. The cpcE and cpcF genes were separately inactivated by insertion of a kanamycin resistance cassette in Synechococcus sp. PCC 7942 to generate mutants R2EKM and R2FKM, respectively, both of which display a substantial reduction in spectroscopically detectable phycocyanin. The levels of beta- and alpha-phycocyanin polypeptides were reduced in the R2EKM and R2FKM mutants although the phycocyanin and linker genes are transcribed at normal levels in the mutants as in the wild type indicating the requirement of the functional cpcE and cpcF genes for normal accumulation of phycocyanin. Two biliprotein fractions were isolated on sucrose density gradient from the R2EKM/R2FKM mutants. The faster sedimenting fraction consisted of intact phycobilisomes. The slower sedimenting biliprotein fraction was found to lack phycocyanin polypeptides, thus no free phycocyanin was detected in the mutants. Characterization of the phycocyanin from the mutants revealed that it was chromophorylated, had a lambda max similar to that from the wild type and could be assembled into the phycobilisome rods. Thus, although phycocyanin levels are reduced in the R2EKM and R2FKM mutants, the remaining phycocyanin seems to be chromophorylated and similar to that in the wild type with respect to phycobilisome rod assembly and energy transfer to the core.
从聚球藻属（Synechococcus sp.）PCC 6301中克隆并测序了两个开放阅读框，分别命名为cpcE和cpcF。cpcE和cpcF基因位于藻胆体杆状操纵子中cpcB2A2基因簇的下游，并且可以独立于上游的cpcB2A2基因簇进行转录。通过在聚球藻属（Synechococcus sp.）PCC 7942中插入卡那霉素抗性盒，分别使cpcE和cpcF基因失活，从而产生突变体R2EKM和R2FKM，这两个突变体在光谱可检测的藻蓝蛋白方面均表现出大幅降低。尽管藻蓝蛋白和连接蛋白基因在突变体中的转录水平与野生型一样正常，但R2EKM和R2FKM突变体中β-和α-藻蓝蛋白多肽的水平降低，这表明功能性的cpcE和cpcF基因对于藻蓝蛋白的正常积累是必需的。从R2EKM/R2FKM突变体中通过蔗糖密度梯度分离出两个双蛋白组分。沉降较快的组分由完整的藻胆体组成。发现沉降较慢的双蛋白组分缺乏藻蓝蛋白多肽，因此在突变体中未检测到游离的藻蓝蛋白。对突变体藻蓝蛋白的表征表明，它已被发色团化，其最大吸收波长与野生型相似，并且可以组装到藻胆体杆中。因此，尽管R2EKM和R2FKM突变体中的藻蓝蛋白水平降低，但剩余的藻蓝蛋白似乎已被发色团化，并且在藻胆体杆组装和向核心的能量转移方面与野生型相似。
