【作者】 张健；

【导师】 周祥山； 张元兴；

【作者基本信息】 华东理工大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 本研究主要分为两部分,紫红曲霉(Monascus purpureus)中PKS基因的克隆与分析；及紫红曲霉MpLigD4基因缺失株的构建。第一部分,本研究使用引物LC1/LC2c得到紫红曲霉中的NR-PKS的基因片段；使用引物LC3/LC5c和KAF2/KAR1得到紫红曲霉中的PR-PKS的基因片段。本研究还创建出一套扩增PKS编码基因的方法,利用此方法,根据ER的氨基酸保守序列设计简并引物,成功获得紫红曲霉中一条ER编码基因的全长核苷酸序列。在此基础上,对该ER基因进行了序列分析,发现该基因在进化上的特异性。这为进一步研究PKS提供了有效的方法,对聚酮类化合物合成的研究具有重要意义。第二部分,使用简并引物得到目的基因片段,通过基因走读的方法得到全长及一定的上下游序列。本研究采用常规双交换的方式缺失MpLigD4基因关键区域(600 bp-1500bp),缺失掉该基因的从上游300 bp到该基因序列上的1700 bp长约2000 bp的序列片段。但是由于其同源重组效率太低,且假阳性太多,所以改进双交换的方法,在打靶质粒同源区的一侧插入oliC31基因。该基因对三乙基锡高度敏感,三乙基锡的使用浓度为10nM,通过改进后的双交换法构建了MpLigD4基因缺失的紫红曲霉菌株。本研究为进一步研究MpLigD4基因功能提供了基础,为今后的基因功能研究提供了基因操作平台。更多还原

【Abstract】 There are two parts of work in this research. Firstly, Clone and analysis the Monascus purpureus PKS genes. Secondly, obtain MpLigD4 disruptant mutant of M. purpureus.Part one, amplified NR-PKS gene fragmant by LC1/LC2c primer pairs and the PR-PKS by LC3/LC5c, KAF2/KAR1 primer pairs. Based on the conserved amino acid sequence codes for ER domain of HR-PKS, a partial ER nucleic acid fragment MpER1 about 300 bp was successfully cloned from M.purpureus by nested PCR, using designed degenerate primers. The analysis of the sequence showed that PKS genes have specific evolution, which provided an effective method to investigate the PKS genes.Part two, partial MpLigD4 nucleic acid fragment was successfully cloned from Monascus purpureus by using designed degenerate primers, and the intact ORF codes for MpLigD4 domian was acquired by Genome walking. Generation of MpLigD4 deletion mutants with the key sequence of MpLigD4 which located 600 bp between 1500 bp replaced by hph gene. Construction of gene-targeting vectors of MpLigD4 which carrying 2-kbp homologous. The hph gene was used as a positive selection marker for transformants, and the oliC31 gene was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. oliC31 codes for an triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. The concentration of triethyltin is 10 nM. Providing MpLigD4-deficient strains as excellent tools for gene targeting in M. purpureus.更多还原