海洋石油降解菌的筛选鉴定及其功能基因研究

Screening and Identification of Oil-Degrading Marine Bacteria and Analysis of Their Functional Genes

以石油原油为唯一碳源,从中国海洋大学海洋微生物菌种库15株菌中筛选到5株石油降解菌。16SrRNA基因测序结果表明,WH072、WH069、WH303、ZXM008和ZXM183分别为耐盐芽孢杆菌(Bacillus halodurans)、除烃海杆状菌(Marinobacter hydrocarbonoclasticus)、弯曲芽孢杆菌(B.flexus)、普里兹湾假交替单胞菌(Pseudoalteromonas prydzensis)和嗜麦芽糖寡养单胞菌(Stenotrophomonas maltophilia)。其中对石油降解率最高的为WH072(高达36.4%),其次是WH069(20.0%)。对这5株菌检测已报道的石油烃降解酶基因,结果在WH072和WH069中均检测到了alkB、P450和almA3种功能基因。根据功能基因编码的氨基酸序列构建系统发育树并分析其各自的进化关系,发现P450基因在一定程度上具有种属特异性。对本研究首次报道的高效石油降解菌耐盐芽孢杆菌WH072,采用高效热不对称性交错PCR(hiTAIL-PCR)方法,成功获得了这3种功能基因的全长序列,其中alkB与已知同源基因序列相似性较低,其结构和功能有待进一步研究。

Crude oil was used as the sole carbon source to screen for petroleum hydrocarbon degrading bacteria from Marine Culture Collection,Ocean University of China（MCCO）.Five strains,WH072,WH069,WH303,ZXM008 and ZXM183,were selected from fifteen strains based on their relative high crude oil degradation rates.Among them,WH072 showed the highest oil degradation rate（up to36.4%）,followed by WH069（20.0%）.They were identified as Bacillus halodurans,Marinobacter hydrocarbonoclasticus,B.flexus,Pseudoalteromonas prydzensis and Stenotrophomonas maltophilia,respectively,based on analysis of 16 SrRNA gene.Genes encoding hydrocarbon-degrading enzymes,including alkB,P450 and almA,were analyzed in these strains.All the three genes were detected in WH072 and WH069.The deduced amino acid sequences of these genes were then used to build a phylogenetic tree with the closest protein sequences from NCBI.The results showed that the phylogeny of gene P450 was in consistent with that of 16 SrRNA gene.B.halodurans WH072 was demonstrated to be involved in oil degradation for the first time.In addition,the ORF sequences of alkB,P450 and almAin B.halodurans WH072 were obtained using high-efficiency thermal asymmetric interlaced PCR（hiTAIL PCR）.Gene alkBin B.halodurans WH072 might encode new alkane hydroxylase and need further studies.