解鸟氨酸拉乌尔菌ZK4菊酯农药降解酶基因BioH的克隆及原核表达

为挖掘新的拟除虫菊酯类农药降解基因，在大肠杆菌中实现异源高效表达，以本课题组前期筛选出的降解谱广泛、降解率较高的菌株解鸟氨酸拉乌尔菌(Raoultella ornithinolytica)为研究对象，克隆其菊酯类农药降解酶基因BioH,在大肠杆菌中表达。通过解鸟氨酸拉乌尔菌全序列进行基因组注释、蛋白注释和通路注释，和已有菊酯类农药降解基因序列比对，筛选出潜在的具有菊酯类农药降解功能的基因；以解鸟氨酸拉乌尔菌基因组DNA为模板，根据功能基因序列设计特异性引物进行PCR扩增，回收目的片段后与pMD19-T载体连接，筛选阳性克隆并鉴定；酶切回收目的片段后和pET32a(+)表达载体连接，转化至大肠杆菌BL21(DE3)中，将验证正确的重组菌经IPTG诱导表达，通过SDS-PAGE分析蛋白表达情况。结果表明：通过筛选得到BioH基因，该基因具有水解酶的作用，同时也具有其他菊酯类农药降解基因序列中存在的保守五肽motif-GXSXG,PCR扩增后得到大小为783 bp的条带，通过重组技术获得阳性克隆，PCR扩增、酶切和DNA测序进行验证，重组菌经0.2 mmol/L IPTG诱导4 h后获得约为...

Cloning and prokaryotic expression of BioH gene of pyrethrin degrading enzyme from Raoulia ornithinolysis ZK4

In order to discover new Tanacetum cinerariifolium like pesticide degradation genes and achieve heterologous and efficient expression in E. coli, the strain Raoultella ornithinolytica with wide degradation spectrum and high degradation rate screened by our research group in the early stage was taken as the research object, and its pyrethroid pesticide degradation enzyme gene BioH was cloned and expressed in E. coli. Through genome annotation, protein annotation and pathway annotation of the whole sequence of R. ornithinolysis, and sequence comparison with existing pyrethroid pesticide degradation genes, potential genes with pyrethroid pesticide degradation function were screened; Using the genomic DNA of R. ornithinolytica as used as a template, and specific primers were designed according to the functional gene sequence for PCR amplification; The target fragment was recovered and connected to the pMD19-T vector, and positive clones were screened and identified; After the target fragment was digested and recovered, it was connected with the pET32a(+)expression vector and transformed into E. coli BL21(DE3). The correct recombinant strain was induced to express by IPTG, and the protein expression was analyzed by SDS-PAGE. The results showed that the BioH gene was obtained by screening, which has the function of hydrolase, and also has the conserved pentapeptide motif-GXSXG in the sequence of other pyrethroid pesticide degradation genes. After PCR amplification, a band with the size of 783 bp was obtained. Positive clones were obtained by recombinant technology. PCR amplification, enzyme digestion and DNA sequencing were verified. The recombinant bacteria obtained about 43 ku(including 6×His tag)is a recombinant protein with molecular weight, which exists in the form of inclusion body. In this experiment, BioH gene was screened and cloned, and the prokaryotic expression vector was successfully constructed and expressed in E. coli BL21(DE3).