Cloning of the gene encoding a protochlorophyllide reductase:: the physiological significance of the co-existence of light-dependent and -independent protochlorophyllide reduction systems in the Cyanobacterium Plectonema boryanum
Cloning of the gene encoding a protochlorophyllide reductase:: the physiological significance of the co-existence of light-dependent and -independent protochlorophyllide reduction systems in the Cyanobacterium Plectonema boryanum
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DOI：
10.1093/oxfordjournals.pcp.a029355
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发表时间：
1998-02-01
影响因子：
4.9
通讯作者：
Hase, T
中科院分区：
文献类型：
--
作者：
Fujita, Y;Takagi, H;Hase, T
蓝藻具有两种原叶绿素还原酶，催化叶绿素向叶绿素的转化，这是叶绿素生物合成途径中的关键步骤;光依赖性（LPOR）和光非依赖性（DPOR）还原酶，我们在蓝细菌Plectonema boryanum基因组DNA的2,131-bp EcoRI片段中发现了一个开放阅读框（ORF 322），由于推导的氨基酸序列与各种植物LPOR的氨基酸序列高度相似，并且在过量表达ORF 322蛋白的大肠杆菌细胞的可溶性组分中检测到LPOR活性，因此将ORF 322定义为Pou基因，其编码的LPOR在P. boryanum中。为研究LPOR的生理意义，采用定向诱变技术，分离到一株LPOR破坏突变体YFP 12，在弱光（10-25 μ E m ~（-2）s ~（-1））条件下，YFP 12与野生型一样生长。但在较高光照条件下（85-130 μ E·m ~（-2）·s ~（-1）），其叶绿素含量显著降低，使其生长受到明显抑制。此外，在最高光强下，YFP 12停止生长并遭受光漂白（170 μ E m（-2）s（-1）），相反，从这些表型特征中，我们得出结论，尽管LPOR和DPOR都有助于在光照下生长的细胞中Chl的合成，LPOR的贡献程度随着光强的增加而增加;没有LPOR，细胞不能在超过130 μ Em（-2）s（-1）的光强下生长。
Cyanobacteria have two protochlorophyllide (Pchlide) reductases catalyzing the conversion of Pchlide to chlorophyllide, a key step in the biosynthetic pathway of chlorophylls (Chls); a light-dependent (LPOR) and a light-independent (DPOR) reductase, We found an open reading frame (ORF322) in a 2,131-bp EcoRI fragment from the genomic DNA of the cyanobacterium Plectonema boryanum, Because the deduced amino acid sequence showed a high similarity to those of various plant LPORs and the LPOR activity was detected in the soluble fraction of Escherichia coli cells over-expressing the ORF322 protein, ORF322 was defined as the pou gene encoding LPOR in P. boryanum. A por-disrupted mutant, YFP12, was isolated by targeted mutagenesiss to investigate the physiological importance of LPOR, YFP12 grew as well as wild type under low light conditions (10-25 mu E m(-2) s(-1)). However, its growth was significantly retarded as a result of a significant decrease in its Chl content under higher light conditions (85-130 mu E m(-2) s(-1)). Furthermore, YFP12 stopped growing and suffered from photobleaching under the highest light intensity (170 mu E m(-2) s(-1)), In contrast, a chlL-disrupted (DPOR-less) mutant YFC2 grew as well as wild type irrespective of light intensity, From these phenotypic characteristics, we concluded that, although both LPOR and DPOR contribute to Chl synthesis in the cells growing in the light, the extent of the contribution by LPOR increases with increasing light intensity; without it, the cells are unable to grow under light intensities of more than 130 mu E m(-2) s(-1).