Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation
Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation
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DOI：
10.1093/abbs/gmu022
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发表时间：
2014-06-01
影响因子：
3.7
通讯作者：
Chen, Hongwen
中科院分区：
文献类型：
--
作者：
Du, Yu;Xie, Guizhen;Chen, Hongwen
pyrG（-）宿主细胞对于基于pyrG（-）的转化系统是必不可少的。通过随机突变分离pyrG（-）宿主细胞受到耗时、遗传背景不清楚和同源重组的潜在干扰的限制。本研究旨在通过pyrG基因缺失的定点突变，构建酿酒曲霉pyrG（-）突变株，为进一步转化奠定基础。利用pMD-pyrGAB载体构建了A.米。3个稳定的pyrG缺失突变体。通过对5-氟乳清酸的抗性分离pyrG，并通过聚合酶链反应分析证实，表明pyrG被完全切除。将Delta pyrG突变体作为pyrG（-）宿主细胞，破坏编码木糖醇脱氢酶的xdh基因，该基因参与A.米。通过转化携带pyrG的pMD-pyrG-xdh破坏质粒，有效地构建了xdh破坏突变体，Delta xdh突变体产生的木糖醇浓度是Delta pyrG受体的3倍。因此，pyrG基因的定点缺失是分离pyrG（-）宿主细胞的有效方法，所建立的宿主-载体系统可用于进一步的功能基因组学分析和A.米。
pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The Delta pyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Delta xdh mutant was three times as much as that of the Delta pyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae.