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噬藻体PaV-LD主要衣壳蛋白、穿孔素和内肽酶基因的克隆及表达分析
CLONING AND EXPRESSION ANALYSIS OF MAJOR CAPSID PROTEIN GENE, ENDOPEPTIDASE AND HOLIN GENE OF CYANOPHAGE PAV-LD

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文摘 最近阐明了水华蓝藻噬藻体PaV-LD(Planktothrix agardhii Virus isolated from Lake Donghu)的全基因组序列,这是一个含有142个ORF的双链DNA噬藻体。在此,我们对其主要衣壳蛋白基因073R,内肽酶和穿孔素基因123L-124L(PaV-LD基因组中两个相邻的ORF)进行了基因克隆与表达分析。将073R克隆后构建原核表达质粒pET-32a-073R,并用IPTG进行诱导表达,073R融合蛋白经纯化后,进行免疫小鼠制备抗体;通过Western blot检测经噬藻体感染宿主细胞后073R的表达时序,结果显示在宿主细胞裂解之初,即PaV-LD感染48h以后073R开始表达,表明073R是一个晚期基因;同时073R推导的氨基酸序列与34株噬藻(菌)体及2株藻病毒(感染真核藻的病毒)的主要衣壳蛋白的氨基酸序列进行序列比对,显示073R与无尾的藻病毒衣壳蛋白亲缘关系更近。PCR扩增内肽酶和穿孔素基因123L-124L,并构建质粒pOP123L-124L,将其转入模式藻集胞藻PCC6803细胞中,质粒pOP123L-124L与藻集胞藻PCC6803基因组发生重组,形成重组藻;测定了重组藻与野生藻的生长速率,并绘制生长曲线;制备超薄切片,进一步比较和观察重组藻与野生藻的超微结构的变化。结果显示重组藻与野生藻存在生长速率与超微形态的显著差异。
其他语种文摘 he 142 open reading frame (ORF) of the genome of cyanophage, PaV-LD (Planktothrix agardhii virus isolated from the Lake Donghu), was elucidated recently. However, the characteristic of genomic, phylogenetic position, and mechanism of infection remained to be examined on PaV-LD, a novel tailless caynophage. With the relatively stable structure and evolutionarily conserved sequence, the major capsid protein (MCP) gene has been an important reference in the classification of these viruses. Endopeptidase and holin were considered to play an important role in the progress of invasion and release of viruses. The MCP gene (073R), endopeptidase gene (123L), and holin gene (124L) of cyanophage PaV-LD were cloned and the expressions were analysed. PaV-LD 073R was amplified and cloned, and the prokaryotic expression plasmid pET-32a-073R was constructed. The prokaryotic expression products of PaV-LD 073R was analyzed by SDS-PAGE after being induced with isopropyl beta-D-thiogalactopyranoside (IPTG). The fusion protein was purified via affinity chromatography and was then used to immunize BALB/C mice to prepare the antiserum against PaV-LD 073R. Next the antiserum was used for Western blotting analysis and the temporal expression pattern of PaV-LD 073R was characterized during PaV-LD infection at different times. The results of western blotting indicated that the MCP was initially expressed at 48h post-infection (p.i.) and developed into particularly enhanced from 60h p.i. to 84h p.i. The data also demonstrated that 073R was a late expression gene of the tailless cyanophage genome. To further investigate the phylogenetic position of PaV-LD, a phylogenetic tree was constructed using the Neighbor-Joining method with 34 tailed cyanophages and 2 phycoviruses (viruses that infect eukaryotic algae). The analysis of the phylogenetic tree revealed that the amino acid sequences in PaV-LD 073R grouped more closely with tailless phycoviruses instead of the tailed cyanophages, indicating that PaV-LD had the morphological characteristics of the tailless phycoviruses. PaV-LD endopeptidase gene and holin gene (two adjacent ORFs in the genome of PaV-LD, 123L-124L) were amplified using polymerase chain reaction (PCR) and the plasmid contained a spectinomycin resistance gene, then the promoter genes pPetE and 123L-124L were constructed and integrated into the genome of Synechocystis sp. PCC6803 via homologous recombination. The growth curves of the recombinant and the wild type of Synechocystis sp. PCC 6803 were drawn, and the growth rate of the recombinant was found to be significantly slower than the wild type. Ultrastructural morphology observation with transmission electron microscopy showed that the cell wall of recombinant was damaged partly or fully, causing protoplast swelling and the leakage of cell contents. Results suggested that PaV-LD 123L-124L could express the functional proteins that played a role in dissolving cell wall and slowing down cell growth in the recombinant Synechocystis sp. PCC 6803.
来源 水生生物学报 ,2013,37(2):252-260 【核心库】
关键词 水华蓝藻噬藻体PaV-LD ; 主要衣壳蛋白基因(073R) ; 内肽酶基因(123L) ; 穿孔素基因(124L)
地址

中国科学院水生生物研究所, 淡水生态与生物技术国家重点实验室, 武汉, 430072

语种 中文
文献类型 研究性论文
ISSN 1000-3207
学科 环境科学基础理论
基金 中国科学院先导专项项目 ;  国家自然科学基金项目 ;  中国科学院水生生物研究所淡水生态与生物技术国家重点实验室基金
文献收藏号 CSCD:4872031

参考文献 共 36 共2页

1.  张奇亚. 浮游病毒. 水生生物学报,2002,26(6):691-696 被引 14    
2.  Sullivan M B. Cyanophages infecting the oceanic cyanobacterium Prochlorococcus. Nature,2003,424(6952):1047-1051 被引 26    
3.  Hambly E. The viriosphere, diversity, and genetic exchange within phage communities. Current Opinion in Microbiology,2005,8(4):444-450 被引 8    
4.  Suttle C A. Marine viruses-major players in the global ecosystem. Nature Reviews Microbiology,2007,5(10):801-812 被引 96    
5.  张奇亚. 一类不可忽视的战略生物资源-淡水与海水中的病毒及其在生态系统中的作用. 中国科学院院刊,2009,24(4):414-420 被引 14    
6.  Liu X. Structural changes in a marine podovirus associated with viral genome release into Prochlorococcus. Nature Structural & Molecular Biology,2010,17(7):830-836 被引 1    
7.  Fischer M G. A virophage at the origin of large DNA transposons. Science,2011,332(6026):231-234 被引 6    
8.  Pope W H. Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution. PloS One,2011,6(1):e16329 被引 5    
9.  Thompson L R. Phage auxiliary metabolic genes and the redirection of cyanobacterial host carbon metabolism. Proceedings of the National Academy of Sciences of the United States of America,2011,108(39):E757-E764 被引 12    
10.  赵以军. 我国首株噬藻体(蓝藻病毒)的分离与鉴定. 自然科学进展,2002,12(9):923-927 被引 23    
11.  Yoshida M. Ecological dynamics of the toxic bloom-forming cyanobacterium Microcystis aeruginosa and its cyanophages in freshwater. Applied and Environmental Microbiology,2008,74(10):3269-3273 被引 15    
12.  高恶斌. 水华蓝藻噬藻体对不同条件培养的宿主细胞感染性分析. 水生生物学报,2012,36(3):420-425 被引 6    
13.  Sabehi G. A novel lineage of myoviruses infecting cyanobacteria is widespread in the oceans. Proceedings of the National Academy of Sciences of the United States of America,2012,109(6):2037-2042 被引 6    
14.  刘露. 一株太湖水域蓝藻噬藻体的分离与鉴定. 水生生物学报,2012,36(2):339-343 被引 4    
15.  刘艳鸣. 武汉东湖浮游病毒的丰度及多样性. 水生生物学报,2005,29(1):1-6 被引 13    
16.  Liu Y M. Seasonal variation of virioplankton in a eutrophic shallow lake. Hydrobiologia,2006,560(1):323-334 被引 14    
17.  Liu Y M. Spatial distribution and morphologic diversity of virioplankton in Lake Donghu, China. Acta Oecologica-International Journal of Ecology,2006,29:328-334 被引 18    
18.  Gao E B. Isolation of a novel cyanophage infectious to the filamentous cyanobacterium Planktothrix agardhii (Cyanophyceae) from Lake Donghu, China. Aquatic Microbial Ecology,2009,54(2):163-170 被引 13    
19.  Gao E B. A Novel Cyanophage with a Cyanobacterial Nonbleaching Protein A Gene in the Genome. Journal of Virology,2012,86(1):236-245 被引 19    
20.  Bowman B R. Structure of the herpesvirus major capsid protein. The EMBO Journal,2003,22(2):757-765 被引 4    
引证文献 4

1 张奇亚 噬藻体生物多样性的研究动态 微生物学通报,2014,41(3):545-559
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2 高恶斌 噬藻体辅助代谢基因(AMGs)研究进展 微生物学通报,2014,41(8):1667-1674
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