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| 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 |
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石鹏格1,傅子莹1,黄泰通1,孙志华2,刘思雨1,秦思轩1,段碧华1*,张力匀1,石生伟1
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| (1.北京农学院 生物与资源环境学院/农业农村部华北都市农业重点实验室, 北京 102206;2.全国畜牧总站, 北京 100125) |
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| 摘要: |
| 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43 ku(包括6×His标签)分子量大小的重组蛋白，该蛋白以包涵体形式存在。本试验筛选并克隆出BioH基因，成功构建原核表达载体，并在大肠杆菌BL21(DE3)中得以表达。 |
| 关键词: 解鸟氨酸拉乌尔菌 菊酯类农药 降解基因 基因克隆 原核表达 |
| DOI：10.11841/j.issn.1007-4333.2023.12.14 |
| 投稿时间：2023-05-28 |
| 基金项目:北京市教委-北京市自然基金联合重点项目(KZ201910020024)；北京市教委项目(KM202210020008)；北京农学院青年教师科研创新能力提升计划(QJKC2022011)；北京农学院学位与研究生教育改革与发展项目(5056516024/002) |
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| Cloning and prokaryotic expression of BioH gene of pyrethrin degrading enzyme from Raoulia ornithinolysis ZK4 |
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SHI Pengge1,FU Ziying1,HUANG Taitong1,SUN Zhihua2,LIU Siyu1,QIN Sixuan1,DUAN Bihua1*,ZHANG Liyun1,SHI Shengwei1
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| (1.Key Laboratory of Urban Agriculture(North China)of Ministry of Agriculture and Rural Affairs/College of Biological and Resource Environment, Beijing College of Agronomy, Beijing 102206, China;2.National Animal Husbandry Station, Beijing 100125, China) |
| Abstract: |
| 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). |
| Key words: Raoulia ornithinolysis pyrethroid pesticide degrading gene gene cloning prokaryotic expression |
