CLONING OF THE CPCE AND CPCF GENES FROM SYNECHOCOCCUS SP PCC-6301 AND THEIR INACTIVATION IN SYNECHOCOCCUS SP PCC-7942
CLONING OF THE CPCE AND CPCF GENES FROM SYNECHOCOCCUS SP PCC-6301 AND THEIR INACTIVATION IN SYNECHOCOCCUS SP PCC-7942
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DOI：
10.1007/bf00039542
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发表时间：
1994-10-01
影响因子：
5.1
通讯作者：
GUSTAFSSON, P
中科院分区：
文献类型：
--
作者：
BHALERAO, RP;LIND, LK;GUSTAFSSON, P
从聚球藻6301中克隆了两个开放阅读框cpcE和cpcF，并进行了序列测定。cpcE和cpcF基因位于藻胆体杆状操纵子中cpcB 2A 2基因簇的下游，并且可以独立于上游cpcB 2A 2基因簇转录。通过在聚球藻属PCC 7942中插入卡那霉素抗性盒分别使cpcE和cpcF基因失活，以分别产生突变体R2 EKM和R2 FKM，这两种突变体均显示出光谱可检测的藻蓝蛋白的显著减少。在R2 EKM和R2 FKM突变体中，β-和α-藻蓝蛋白多肽的水平降低，尽管藻蓝蛋白和接头基因在突变体中以与野生型中相同的正常水平转录，表明藻蓝蛋白的正常积累需要功能性cpcE和cpcF基因。在蔗糖密度梯度上从R2 EKM/R2 FKM突变体中分离出两种胆蛋白组分。快速沉降的部分由完整的藻胆体组成。发现较慢沉降的胆蛋白级分缺乏藻蓝蛋白多肽，因此在突变体中没有检测到游离藻蓝蛋白。从突变体的藻蓝蛋白的表征表明，它是发色团，具有类似于从野生型的λ（max），并可以组装成藻胆体杆。因此，尽管藻蓝蛋白水平在R2 EKM和R2 FKM突变体中降低，但剩余的藻蓝蛋白似乎是发色团化的，并且在藻胆体杆组装和能量转移到核心方面与野生型相似。
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.