柴油降解基因工程菌的构建及降解特性

发布时间：2018-03-11 11:53

本文选题：柴油　切入点：降解　出处：《微生物学通报》2017年06期 　论文类型：期刊论文

【摘要】：【目的】构建柴油降解基因工程菌,提高柴油降解速率,研究p450基因在柴油降解过程中的作用。【方法】将Alcanivorax borkumensis SK2的p450基因合成后,连接至烷烃响应表达载体p Com8中,构建该基因的表达载体p450-SK2/p Com8,并将其转入大肠杆菌DH5α中,通过SDS-PAGE检测该基因在大肠杆菌DH5α中的表达,并将重组质粒p450-SK2/p Com8转入柴油降解菌Acinetobacter sp.Y9中,构建基因工程菌p450-SK2/Y9,研究工程菌p450-SK2/Y9对柴油的降解特性及p450基因在构建的工程菌p450-SK2/Y9中的表达。【结果】PCR、酶切及测序结果表明重组质粒p450-SK2/p Com8构建正确。当柴油诱导浓度大于1%时,目的基因在大肠杆菌DH5α中的蛋白表达量较大,且随着诱导时间的延长而呈增加趋势。通过PCR检测构建的基因工程菌p450-SK2/Y9中的p450基因表明,工程菌构建正确,利用单菌株降解柴油时,宿主菌Y9与工程菌p450-SK2/Y9的柴油降解效率未见明显差异,但工程菌p450-SK2/Y9在构建的菌群中对柴油降解的促进效果明显。SDS-PAGE结果表明,p450基因在构建的工程菌p450-SK2/Y9中能得到准确表达,在混合菌中的表达量高于单菌株。【结论】柴油降解基因工程菌在混合菌群中对柴油降解具有促进作用,而在单菌株情况下未见促进作用,且p450基因的蛋白表达在混合菌中也高于单菌株,这对于提高柴油的降解速率及研究p450基因在柴油降解过程中的作用机理具有一定意义。
[Abstract]:[objective] to construct genetically engineered bacteria for diesel oil degradation, to improve diesel degradation rate, and to study the role of p450 gene in diesel oil degradation. [methods] the p450 gene of Alcanivorax borkumensis SK2 was synthesized and ligated into the alkane response expression vector p Com8. The expression vector p450-SK2 / p Com8 was constructed and transferred into Escherichia coli DH5 伪. The expression of the gene in E. coli DH5 伪 was detected by SDS-PAGE, and the recombinant plasmid p450-SK2 / p Com8 was transformed into Acinetobacter sp.Y9. The biodegradation of diesel oil by p450-SK2 / Y9 and the expression of p450 gene in p450-SK2 / Y9 were studied. [results] PCR, restriction endonuclease digestion and sequencing showed that the recombinant plasmid p450-SK2 / P Com8 was constructed correctly. The expression of p450 gene in Escherichia coli DH5 伪 was higher than that in Escherichia coli DH5 伪, and increased with the prolongation of induction time. The p450 gene in p450-SK2 / Y9 genetically engineered strain constructed by PCR showed that the construction of the engineered strain was correct. When the single strain was used to degrade diesel oil, there was no significant difference in diesel degradation efficiency between the host strain Y9 and the engineering strain p450-SK2 / Y9. The results of SDS-PAGE showed that the gene of p450-SK2 / Y9 could be expressed accurately in the constructed strain p450-SK2 / Y9. [conclusion] the gene engineering bacteria of diesel degradation can promote the degradation of diesel oil in the mixed bacteria, but not in the case of a single strain, but not in the case of a single strain. [conclusion] the gene engineering bacteria can promote the degradation of diesel oil in the mixed bacteria, but not in the case of a single strain. The protein expression of p450 gene is also higher in mixed bacteria than in single strain, which is of significance for improving the degradation rate of diesel oil and studying the mechanism of p450 gene in the degradation of diesel oil.
【作者单位】：海军医学研究所;
【分类号】：X172

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