橙色红曲菌pyrG缺陷株的筛选及鉴定

Screening and Identification of Orange Monascus Aurantiacus pyrG Mutant

[目的]筛选和鉴定橙色红曲菌pyrG缺陷株。[方法]以橙色红曲菌AS3.4384(Monascus aurantiacus)为出发菌株,运用紫外照射致基因突变,随后对尿嘧啶依赖型菌株的pyrG基因进行测序比对,筛选pyrG基因突变株,最后用含有米曲霉pyrG基因的pAOP互补质粒对其进行基因转化试验。[结果]经紫外诱变得到一株尿嘧啶依赖型的5-FOA抗性菌株UM28。扩增其pyrG基因并进行测序,发现UM28的pyrG基因在核苷酸序列+220bp的位置发生了突变,进而导致氨基酸水平上Pro→Ser的突变,造成了乳清苷-5'-磷酸脱羧酶的失活。UM28通过基因转化能够接受含有米曲霉pyrG基因的互补质粒恢复野生表型,且回复突变率低于10-8,能够用于红曲菌同源转化系统的构建。[结论]获得了一株红曲菌pyrG基因缺陷株UM28,它可被用作基因工程的受体菌。

[Objective]The research aimed to screen and identify the Monascus aurantiacus pyrG mutant.[Method]Took orange Monascus AS3.4384 （Monascus aurantiacus） as the starting strain,given rise to genic mutation with UV mutagenesis,then the pyrG gene of uracil-dependent strain was sequenced and compared,the pyrG gene mutants was screened,at last,we used pAOP complementary plasmid based on the pyrG gene of Aspergillus oryzae to carry out the gene transformation experiment.[Result] A pyrG mutant strain from M.aurantiacus AS3.4384,named UM28,was isolated by resistance to 5-fluoroorotic acid （5-FOA） after UV mutagenesis.Sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position ＋ 220,caused substitution of Pro→Ser,with inactivation of OMPdecase function.Complementation of M.aurantiacus pyrG mutant was performed by transforming Aspergillus oryzae pyrG gene into UM28,and it transformed UM28 into uridine prototrophy.UM28 was a uridine auxotroph with a frequency of reverse mutation of less than 10-8,and it could be used in the Monascus homologous transformation system.[Conclusion]A strain of the Monascus pyrG mutant UM28 was obtained,which can be used as the recipient strain of genetic engineering.