一株石油烃降解菌TY22烷烃羟化酶基因的克隆及功能研究

Cloning and Functional Analysis of Alkane Hydroxylase Gene of a Petroleum Hydrocarbon Degrading Bacteria TY22

doi:
摘要:
[目的]对一株石油烃降解菌TY22的烷烃羟化酶基因alkB克隆测序,并对其外源表达和功能进行研究.[方法]PCR扩增获得alkB基因部分序列,再根据该序列通过染色体步移技术,最终获得完整CDS序列.将alkB基因与pET21b(+)载体构建重组质粒,并转入E.coli BL21(DE3)进行表达.再将alkB基因克隆至pCom8载体中,分别转入E.coli GEc137(pGEc47△B)和Pseudomonas fluorescens KOB2Δ1进行基因功能的互补试验.[结果]染色体步移获得了1236 bp的烷烃羟化酶基因完整CDS序列(GenBank Accession:KU867870).诱导表达发现,重组菌在0.1 mmol/L IPTG、30℃条件下诱导培养6 h的表达效果最佳,得到与预期相符的46 kDa蛋白.基因功能互补试验发现,重组菌能在以C12~C16为唯一碳源的培养基中生长.[结论]TY22菌株的烷烃羟化酶基因完整CDS序列全长1236 bp,能氧化C12~C16的中长链烷烃,并能在大肠杆菌中有效表达.
Abstract:
[Objective] To clone and sequence the alkane hydroxylase gene alkB of a petroleum hydrocarbon degrading bacteria TY22,then to clone alkB into express vector for expressing and identifying its function.[Method] First,use PCR amplification to get partial sequence of 1236 bp alkB gene,then obtain the complete CDS sequence by chromosome walking techniques.Second,clone alkB gene into pET21b( +) vector,and transform the recombinant plasmid into E.coli BL21(DE3) for expressing.Third,clone alkB gene into pCom8 vector,and transform the recombinant plasmid into E.coli GEc137(pGEc47△B) and Pseudomonas fluorescens KOB2Δ1 respectively for gene function complementation test.[Result] We got the complete CDS sequence of 1236 bp alkane hydroxylase gene through chromosome walking techniques ( GenBank Accession:KU867870).The expression strain had the most expression with 0.1 mmol/L IPTG,30 ℃culture for 6 hours,relative molecular mass 46 kDa was also in line with expectation.Gene function complementation test showed that the recombinant strains can grow in the medium with n-alkanes C12~C16 as sole carbon source.[Conclusion] Alkane hydroxylase gene alkB complete CDS sequence length of strain TY22 was 1236 bp.It can oxidize n-alkanes with chain length C12 ~C16 ,and efficient expression in E.coli BL21(DE3).
作者 唐赟    敬珊    邹书珍    冀黎骏    杨雪    余小丽
Author:
作者单位 西华师范大学生命科学学院,四川南充,637009
刊 名: 安徽农业科学   ISTICPKU
Journal: Journal of Anhui Agricultural Sciences
年,卷(期) 2017, 45(36)
分类号 S188
关键词: 石油烃降解菌 知识脉络    烷烃羟化酶 知识脉络    表达 知识脉络    功能 知识脉络   
Keywords: Petroleum hydrocarbon degrading bacteria    Alkane hydroxylase    Expression    Function   
机标分类号
基金项目 四川省科技厅应用基础研究项目,四川省南充市科技局重点项目
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