环境基因组DNA脂肪酶基因克隆与分析

Cloning and Analysis of a Lipase Gene from Environmental Genome DNA

为了建立从环境基因组DNA中直接克隆脂肪酶基因的通用方法,抽提餐馆灶台油污的环境基因组DNA,采用简并PCR等分子技术从中克隆到脂肪酶基因lip42及其对应的全长cDNA.序列分析表明该基因的cDNA编码区序列全长为1692bp,编码1段由19个氨基酸残基组成的信号肽和1段由544个氨基酸残基组成的成熟肽,计算相对分子质量为61541.51,其中含有强碱性氨基酸残基42个,强酸性氨基酸残基56个,等电点pI为5.428,有2个可能的N-糖基化位点.BLAST比对分析表明该序列与已发表的白地霉(Geotrichum geotrichum)脂肪酶基因(U02541)在核苷酸水平的一致性为99%.该基因序列提交GenBank,登录号为DQ313172.将该脂肪酶基因的开放阅读框克隆到毕赤酵母表达载体,转化毕赤酵母GS115,经罗丹明B平板功能筛选得到具脂肪酶活性的重组毕赤酵母菌株[GS115(pAMB768)],验证了直接从环境基因组DNA克隆脂肪酶基因的有效性.

A lipase gene was cloned by degenerate PCR from environmental genome DNA extracted from the oil dirt on the hearth of a restaurant and designated as lip42.Its sequence was submitted to GenBank with the accession number of DQ313172 obtained.The cDNA of the putative lipase gene （lip42） consisted of 1 692 bp,including an open reading frame encoding a 19-amino acid signal peptide at the N-terminal end and a 544-amino acid mature protein with a predicted molecular mass of 61 541.51,with pI value of 5.482 and 2 potential N-glycosylation sites （N-X-T/S）.The open reading frame was transformed into Pichia pastoris strain GS115 under the control of the AOX1 promoter by using vector pHBM906.Secretion expression of lip42 was detected from P.pastoris recombinant strain [GS115 （pAMB768）].Fig 3,Tab 1,Ref 27