一株石油烃降解菌TY22烷烃羟化酶基因的克隆及功能研究

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(p GEc47△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1 236 bp的烷烃羟化酶基因完整CDS序列(Gen Bank 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1 236 bp,能氧化C12~C16的中长链烷烃,并能在大肠杆菌中有效表达。

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

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).