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葛仙米藻蓝素铁氧还蛋白还原酶基因Ns-PcyA的克隆、表达及其分子结构基础
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引用本文:汪海洋,朱格格,卢 婵,程 超,李 伟,方 庆.葛仙米藻蓝素铁氧还蛋白还原酶基因Ns-PcyA的克隆、表达及其分子结构基础[J].西北农业学报,2021,(12):1863~1870
DOI:10.7606/j.issn.1004-1389.2021.12.012
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作者单位
汪海洋,朱格格,卢 婵,程 超,李 伟,方 庆 (1.湖北民族大学 生物科学与技术学院,湖北恩施 445000
2.生物资源保护与利用湖北省重点实验室,湖北恩施 445000) 
基金项目:湖北省教育厅团队项目(T201312);国家自然科学基金(31560434);湖北省重点实验室开放基金(PKLHB1916)。
中文摘要:为研究葛仙米藻蓝胆素的细胞内合成作用,采用聚合酶链式反应(PCR)、构建融合His标签重组载体和SDS-PAGE电泳等试验方法,克隆与表达葛仙米藻蓝素铁氧还蛋白还原酶基因Ns-PcyA,并对其蛋白高级结构进行模拟分析。结果显示,葛仙米藻蓝素铁氧还蛋白还原酶基因Ns-PcyA密码区全长744 bp,预测编码一含247个氨基酸的蛋白质肽链。Ns-PcyA与同属其他藻种中的同源蛋白氨基酸序列总相似性可达 95.66%,并且富含半胱氨酸(7个Cysteine残基)。以点型念珠藻(Nostoc punctiforme)为参照,葛仙米Ns-PcyA蛋白多肽链除C末端多两个氨基酸外,还出现8个位点氨基酸的显著替换;分子进化显示, Ns-PcyA很可能源于点型念珠藻的同源基因,而与发状念珠藻和普通念珠藻中PcyA基因形成明显分支。这表明PcyA基因的生物学功能在不同念珠藻中可能产生变异。构建Ns-PcyA基因的重组表达载体,并在大肠杆菌中成功表达分子量近29.0 ku的目的蛋白。高级结构模拟显示,Ns-PcyA单分子中主要包含α螺旋和β折叠片两类二级结构,α螺旋和β折叠片进一步形成类似三面夹心式高级结构。其外围两侧共分布5个较典型的α螺旋,而8个反向平行β折叠片夹于其中,形成一疏水内核。本研究结果为深入了解和开发葛仙米藻蓝胆素的细胞内合成作用奠定了试验基础。
中文关键词:藻蓝蛋白  藻蓝素铁氧还蛋白还原酶  葛仙米  克隆  结构
 
Cloning, Expression and Advanced Molecular Structural Basis of Phycocyanin Ferredoxin Reductase Gene Ns-PcyA in Nostoc sphaeroides Küzting
Abstract:To explore the phycocyanobilin biosynthesis in Nostoc sphaeroides, PCR and other experimental methods such as construction of recombination vector of fusion His tag and SDS-PAGE were used to study the cloning and expression of the phycocyanin ferredoxin reductase gene Ns-PcyA.The results showed that the total length PcyA in the coding region was 744 bp, which predicted to deduce a polypeptide chain containing 247 amino acids.Compared the amino acid sequences of PcyA homologous proteins in several other phycophytes, the total similarity was 95.66%.With the Nostoc punctiforme as a reference, in addition to two more amino acids at the C-terminal, there was a significant substitution of amino acids at eight sites; molecular evolution showed that the Ns-PcyA probably originated from Nostoc punctiforme homologous protein,while it formed distinct branches with PcyA proteins in Nostoc flagelliforme and Nostoc commune, indicating that the biological function of the PcyA protein may vary in different Nostoc species.We successfully constructed recombinant expression vectors of Ns-PcyA enzyme gene and successfully expressed target protein of around 29.0 ku in E.coli.Ns-PcyA single molecule could form a binder-like structure, in which five typical α helices were distributed on both sides of the periphery, while eight β sheets were sandwiched in the relatively closed space formed by the helix on both sides.Taken together, the results provide experimental basis for further exploration of phycocyanobilin in Nostoc sphaeroides.
keywords:Phycocyanin  Phycocyanobilin ferridoxin reductase  Nostoc sphaeroides Kützing  Cloning  Structure
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