螺旋藻藻蓝蛋白裂合酶cpcE-F基因的克隆、表达及进化分析
摘要
螺旋藻是一种具有重要经济价值和开发前景的原核生物,关于螺旋藻的分类问题,曾经有一段争论,由于目前越来越多的人把螺旋藻与全价营养和多种医疗功能结合在一起,因此其分类显得更为重要。螺旋藻的传统分类是介于其表现型,但是表现型容易受到环境的影响,这给螺旋藻的分类带来了不小的麻烦;随着分子技术的飞速发展,许多基因被用来进行螺旋藻的系统分析,并取得一定成果。藻胆蛋白是一类结构相似的色素蛋白,它们是由藻胆色素与相应的脱辅基蛋白以硫醚键共价结合而成的,裂合酶是催化该连接反应的酶,藻蓝蛋白是藻胆蛋白中的一种。裂合酶cpcE和cpcF基因最早在聚球藻Synechococcus sp.PCC7002中发现和鉴定,该基因在不同的藻种中具有较高的同源性,适宜作为分子进化的工具,使用该基因作为进化分析的报道还没有。目前,对于裂合酶分析较多的是对α亚基的研究,并且对于螺旋藻自身的藻蓝蛋白α亚基的催化研究还没有报道。
本文通过GenBank中报道的其他蓝藻关于裂合酶cpcE和cpcF基因设计引物,进行PCR扩增,首次从螺旋藻FACHB314和盐泽螺旋藻FACHB351中克隆得到了cpcE-F裂合酶基因全序列,并对其进行了序列分析和进化研究。结果表明:裂合酶cpcE-F基因的GC含量在Spirulina platensis FACHB314和Spirulina subsalsa FACHB351中分别为50.5%和50.6%,在已经报道的蓝藻cpcE-F基因中处于上游水平,该基因的GC%含量在螺旋藻各个藻种间存在差异,螺旋藻FACHB314 cpcE-F基因与已经报道螺旋藻和节旋藻核苷酸序列的相似性分别为95.0%和94.8%、氨基酸序列的相似性分别为93.7%和93.3%;盐泽螺旋藻FACHB351该基因与已经报道螺旋藻和节旋藻核苷酸序列的相似性分别为95.0%和94.8%、氨基酸序列的相似性分别为94.7%和95.1%,能够使用cpcE-F基因将螺旋藻和节旋藻以及其他蓝藻区分开。同时,我们还对得到的cpcE-F基因进行了二级结构的预测分析,螺旋藻FACHB314和盐泽螺旋藻FACHB351中cpcE-F基因的Mw分别为119587.0kDa和119576.0kDa,等电点分别为5.76和5.69,其蛋白质的不稳定系数分别为23.12和22.92。分子生物学的方法对蓝藻进行快速准确地分析是非常必要的,裂合酶cpcE-F基因位点提供了快速直接的鉴定的方法,本研究首次使用裂合酶基因为螺旋藻的分类提供分子生物学证据。
通过构建表达质粒,我们证实、鉴定了该裂合酶基因。其在大肠杆菌中的表达研究表明:从Spirulina platensis FACHB314和Spirulina subsalsa FACHB351克隆的得到的藻蓝蛋白裂合酶cpcE和cpcF基因结构完整,能够编码表达裂合酶CpcE和CpcF起到催化藻胆色素与脱辅基藻蓝蛋白结合的作用,也首次证实了螺旋藻藻蓝蛋白α亚基自身的催化作用。
Spirulina is a high economic value prokaryote whose exploitation has a bright future. It is usually argued about the classification. Due to the connection of its nourishment and medical value, the classification of Spirulina appares to be very important. The conventional classification of it is base on the phenotype, but the phenotype is influenced by the surrounding, therefore the difficult appears. By the usage of molecular technique, its classification develops rapidly, and the results of that are obvious. Biliproteins are a wide spread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. It is catalyzed by the lyase. The cpcE and cpcF lyase genes are found in the cyanobacterium Synechococcus sp. PCC 7002. The function of them has been proved. We find that the cpcE and cpcF genes have homology in different cyanobacterias. They are fit for phyletic evolution analysis. At present we have not found the new report about phyletic evolution analysis by this genes. We also have not found the new report about the catalytic reaction by this gene in Spirulina itself.
The complete sequence of lyase genes were obtained by PCR. Primers were designed according to the published gene sequences of cpcE-F. The sequences are also totally 1488bp. We firstly obtain the sequences of cpcE-F in Spirulina platensis and Spirulina subsalsa. The GC contents of cpcE-F are 50.6% and 50.5%. The similarities of nucleotide in FACHB314 are different in 95.0% and 94.8%, amino acid are different in 93.7% and 93.3%, The similarities of nucleotide in FACHB351 are different in 95.0% and 94.8%, amino acid are different in 94.7% and 95.1%. Mw are 119587.0kDa and 119576.0kDa, pI are 5.76 and 5.69, the aliphatic index are 23.12 and 22.92. We can reach the conclusion that the lyase gene should be use to make a difference in Spirulina.
To verify the function of lyase, we have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to theα-subunit of apophycocyanin at the appropriate site,α-Cys-84, to form the correct adduct. We also firstly suggest that CpcE and CpcF of Spirulina could catalyse the connection of PCB to phycocyaninαsubunit in vivo, and therefore obtained the transformation activity of light energy.
本文通过GenBank中报道的其他蓝藻关于裂合酶cpcE和cpcF基因设计引物,进行PCR扩增,首次从螺旋藻FACHB314和盐泽螺旋藻FACHB351中克隆得到了cpcE-F裂合酶基因全序列,并对其进行了序列分析和进化研究。结果表明:裂合酶cpcE-F基因的GC含量在Spirulina platensis FACHB314和Spirulina subsalsa FACHB351中分别为50.5%和50.6%,在已经报道的蓝藻cpcE-F基因中处于上游水平,该基因的GC%含量在螺旋藻各个藻种间存在差异,螺旋藻FACHB314 cpcE-F基因与已经报道螺旋藻和节旋藻核苷酸序列的相似性分别为95.0%和94.8%、氨基酸序列的相似性分别为93.7%和93.3%;盐泽螺旋藻FACHB351该基因与已经报道螺旋藻和节旋藻核苷酸序列的相似性分别为95.0%和94.8%、氨基酸序列的相似性分别为94.7%和95.1%,能够使用cpcE-F基因将螺旋藻和节旋藻以及其他蓝藻区分开。同时,我们还对得到的cpcE-F基因进行了二级结构的预测分析,螺旋藻FACHB314和盐泽螺旋藻FACHB351中cpcE-F基因的Mw分别为119587.0kDa和119576.0kDa,等电点分别为5.76和5.69,其蛋白质的不稳定系数分别为23.12和22.92。分子生物学的方法对蓝藻进行快速准确地分析是非常必要的,裂合酶cpcE-F基因位点提供了快速直接的鉴定的方法,本研究首次使用裂合酶基因为螺旋藻的分类提供分子生物学证据。
通过构建表达质粒,我们证实、鉴定了该裂合酶基因。其在大肠杆菌中的表达研究表明:从Spirulina platensis FACHB314和Spirulina subsalsa FACHB351克隆的得到的藻蓝蛋白裂合酶cpcE和cpcF基因结构完整,能够编码表达裂合酶CpcE和CpcF起到催化藻胆色素与脱辅基藻蓝蛋白结合的作用,也首次证实了螺旋藻藻蓝蛋白α亚基自身的催化作用。
Spirulina is a high economic value prokaryote whose exploitation has a bright future. It is usually argued about the classification. Due to the connection of its nourishment and medical value, the classification of Spirulina appares to be very important. The conventional classification of it is base on the phenotype, but the phenotype is influenced by the surrounding, therefore the difficult appears. By the usage of molecular technique, its classification develops rapidly, and the results of that are obvious. Biliproteins are a wide spread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. It is catalyzed by the lyase. The cpcE and cpcF lyase genes are found in the cyanobacterium Synechococcus sp. PCC 7002. The function of them has been proved. We find that the cpcE and cpcF genes have homology in different cyanobacterias. They are fit for phyletic evolution analysis. At present we have not found the new report about phyletic evolution analysis by this genes. We also have not found the new report about the catalytic reaction by this gene in Spirulina itself.
The complete sequence of lyase genes were obtained by PCR. Primers were designed according to the published gene sequences of cpcE-F. The sequences are also totally 1488bp. We firstly obtain the sequences of cpcE-F in Spirulina platensis and Spirulina subsalsa. The GC contents of cpcE-F are 50.6% and 50.5%. The similarities of nucleotide in FACHB314 are different in 95.0% and 94.8%, amino acid are different in 93.7% and 93.3%, The similarities of nucleotide in FACHB351 are different in 95.0% and 94.8%, amino acid are different in 94.7% and 95.1%. Mw are 119587.0kDa and 119576.0kDa, pI are 5.76 and 5.69, the aliphatic index are 23.12 and 22.92. We can reach the conclusion that the lyase gene should be use to make a difference in Spirulina.
To verify the function of lyase, we have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to theα-subunit of apophycocyanin at the appropriate site,α-Cys-84, to form the correct adduct. We also firstly suggest that CpcE and CpcF of Spirulina could catalyse the connection of PCB to phycocyaninαsubunit in vivo, and therefore obtained the transformation activity of light energy.
引文
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