偏肿革裥菌Lg-mnp1基因克隆及表达研究

[目的]研究偏肿革裥菌Lg-mnp1基因的克隆及表达情况.[方法]根据白腐菌锰过氧化物酶（MnP）基因保守序列设计引物,采用PCR、RACE及染色体步移等方法,克隆偏肿革裥菌Lenzites gibbosa CB1的全长MnP1基因（GenBank登录号JQ327834）,进行蛋白序列分析,并通过双酶切的方法获得重组质粒,将该重组质粒电转化至巴斯德毕赤酵母中进行异源表达.[结果]获得偏肿革裥菌Lenzites gibbosa CB1的全长MnP1基因,命名为Lg-mnp1.蛋白序列分析表明Lg-MnP1含有4个二硫键属于短MnP,预测成熟蛋白分子量为35.94kD,pI为4.43.通过双酶切的方法将Lg-mnp1的开放阅读框（ORF）序列连接到pPICZB载体上,获得重组质粒pPICZB/%-mnp1,转化子经PCR验证后,在BMMY培养基中甲醇诱导培养,在未添加血红素的培养液中MnP胞外酶活在72 h达到最高为7 U/L,表明Lg-mnp1已被转化至毕赤酵母中并可诱导表达.[结论]该方法对偏肿革裥菌Lg-mnp1基因的克隆及表达情况进行研究,为偏肿革裥菌Lg-mnp1基因的进一步应用提供了依据.

Cloning of Lg-mnp1 Gene from Lenzites gibbosa and Heterologous Expression

[Objective] To study cloning and expression of Lg-mnp1 gene from Lenzites gibbosa.[Method] A full length DNA gene Lg-mnp1 encoding for an isoenzyme of manganese peroxidase （MnP） from Lenzites gibbosa stain CB1 （GenBank accession No.JQ327834） was cloned by the methods of PCR,RACE,and Genome Walking PCR with primers designed based on the conserved sequences of the known MnP genes from other white-rot fungi and amplified DNA fragments.[Result] Protein sequence analysis showed that Lg-MnP1 belonged to short MnP due to its 4 disulfide bonds.The predicted molecular mass of mature Lg-MnP1 was 35.94 kDa,and the pI was 4.43.The full open reading frame（ORF） sequence of Lg-mnp1 was cloned by restriction enzyme digestion primers.The recombinant plasmid pPICZB/Lg-mnp1 was constructed by ligating the ORF sequence and the expression plasmid pPICZB both digested by the restriction enzyme EcoRI and XbaI.Then the pPICZB/Lg-mnp1 was transformed into P.pastoris by electroporation.After verified by PCR,two transformant Pichia strains were cultured in BMMY medium and induced by anhydrous methanol added at each 24 h,and their MnP activities were determined during 96 h.Results showed that the transformant strain 1 could produce MnP activity in the absence of heme,and the highest MnP activity in the culture supernatant was 7 U/L after 72 h incubation,indicating that the gene Lg-mnp1 had been effectively transformed into the recombinant Pichia pastoris SMD1168H-Lg-mnp1 and expressed in inductive environment.[Conclusion] The cloning and expression of Lg-mnp1 gene from Lenzites gibbosa was studied,which will provide a reference for further application of Lg-mnp1 gene.