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首页> 外文期刊>Archives of microbiology >Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A.
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Cloning and expression of a toxin gene from Pseudomonas fluorescens GcM5-1A.

机译：荧光假单胞菌GcM5-1A毒素基因的克隆和表达。

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Pseudomonas fluorescens GcM5-1A was isolated from the pine wood nematode (PWN), Bursaphelenchus xylophilus, obtained from wilted Japanese black pine, Pinus thumbergii, in China. In this paper, a genomic library of the GcM5-1A strain was constructed and a toxin-producing clone was isolated by bioassay. Nucleotide sequence analysis revealed an open reading frame of 1,290 bp encoding a protein of 429 amino acids with N-terminal putative signal peptide of 36 amino acids, which shared a similarity of 83, 82 and 80% identity with hypothetical protein PFLU2919 from P. fluorescens SBW25, Dyp-type peroxidase family protein from P. fluorescens Pf-5 and Tat-translocated enzyme from P. fluorescens Pf0-1, respectively. The gene encoding a full-length protein or without the putative signal peptide was cloned and expressed as a soluble protein in E. coli. The recombinant protein was purified to electrophoretic homogeneity by affinity chromatography using a Ni2+ matrix column. Its relative molecular weight was estimated to be 48.5 kDa by SDS-PAGE for full-length protein, and 45.0 kDa for the recombinant protein without putative signal peptide. Bioassay results showed that the recombinant protein with or without the putative signal peptide was toxic to both suspension cells and P. thunbergii seedlings. HPLC analysis demonstrated that components in branch extracts of P. thunbergii were significantly changed after addition of the recombinant full-length protein and hydrogen peroxide, which indicated that it is probably a peroxidase. This study offers information that can be used to determine the mechanism of pine wilt disease caused by the PWN.
机译：荧光假单胞菌GcM5-1A分离自松木线虫（PWN），从中国枯萎的日本黑松Pusus thumbergii获得。在本文中，构建了GcM5-1A菌株的基因组文库，并通过生物测定法分离了产生毒素的克隆。核苷酸序列分析揭示了一个1,290 bp的开放阅读框，其编码429个氨基酸的蛋白质与N端推定的信号肽具有36个氨基酸，与荧光假单胞菌的假定蛋白质PFLU2919具有83％，82％和80％的相似性。 SBW25，荧光假单胞菌Pf-5的Dyp型过氧化物酶家族蛋白和荧光假单胞菌Pf0-1的Tat易位酶。克隆了编码全长蛋白质或不含推定信号肽的基因，并在大肠杆菌中表达为可溶性蛋白质。通过使用Ni2 +基质柱的亲和色谱将重组蛋白纯化至电泳均一。通过全长SDS-PAGE估计其相对分子量为48.5 kDa，对于不含推定信号肽的重组蛋白，其相对分子量为45.0 kDa。生物测定结果表明，带有或不带有推定信号肽的重组蛋白对悬浮细胞和P. thunbergii幼苗均具有毒性。 HPLC分析表明，在加入重组全长蛋白和过氧化氢后，雷氏假单胞菌分支提取物中的成分发生了显着变化，这表明它可能是一种过氧化物酶。这项研究提供的信息可用于确定由PWN引起的松萎病的机制。

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