橙色红曲菌中基于pyrG标记的同源转化系统的建立
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摘要
本文采用CODEHOP策略设计简并引物,从橙色红曲菌(Monascus aurantiacus)的基因组DNA中克隆得到pyrG基因片段,然后运用PCR法筛选红曲菌Fosmid文库,获得了pyrG基因全长。以橙色红曲菌AS3.4384为出发菌株,经紫外诱变获得尿嘧啶依赖型的5-FOA抗性菌株,经测序和基因转化试验鉴定出UM28为pyrG缺陷株。对PEG介导的转化方法优化后建立了以pyrG为筛选标记的红曲菌同源转化系统,并构建了含有GFP蛋白的表达载体pGFP-pyrG,通过基因转化试验,在红曲菌中以pyrG为筛选标记进行了GFP蛋白的表达。主要研究内容如下：
1.根据已报道的真菌乳清苷-5'-磷酸脱羧酶基因(pyrG)的氨基酸序列,采用CODEHOP策略设计简并引物,从橙色红曲菌的基因组DNA中克隆得到pyrG基因片段。然后运用PCR法筛选红曲菌Fosmid文库,获得了pyrG基因全长(GenBank登录号：CU723506)。经过blastx比较发现,红曲菌pyrG基因与Aspergillus flavus NRRL3357的氨基酸序列同源性最高,达到81%。该基因能够作为筛选标记应用于红曲菌同源转化系统；
2.以橙色红曲菌AS3.4384为出发菌株,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+220 bp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-,能够用于红曲菌同源转化系统的构建；
3.对UM28菌株的原生质体制备、再生和转化方法进行了优化,采用原生质体PEG介导转化将互补质粒pAOP和pMAP分别转入UM28中,能够使其恢复野生表型。建立了以pyrG为筛选标记的红曲菌同源转化系统；
4.通过DNA重组技术构建了表达载体pGFP-pyrG,载体中含有pyrG筛选标记和GFP蛋白表达单元。通过基因转化试验,在红曲菌中以pyrG为筛选标记进行了GFP蛋白的表达。
In this paper, pyrG gene fragment was cloned from Monascus aurantiacus genomic DNA using degenerate primers designed with CODEHOP strategy. And its complete sequence was obtained by a PCR-based method for screening fosmid library. Uridine auxotrophy strains from M. aurantiacus AS3.4384 were isolated by resistance to 5-FOA after UV mutagenesis. One positive colony was obtained and named UM28, it was further confirmed by sequencing and transformation. The formation of protoplast condition and the regeneration of protoplast condition of UM28 strain were optimized, and a homologous transformation system using pyrG as a selection marker of UM28 is established. A plasmid pGFP-pyrG containing the GFP expression cassette was constructed and transformed into UM28 protoplast, expression of transformed gene was evaluated using the GFP gene. The major findings were expounded as follows:
1. According to the known Orotidine-5'-phosphate decarboxylase, pyrG gene fragment was cloned from Monascus aurantiacus genomic DNA using degenerate primers designed with CODEHOP strategy. And its complete sequence was obtained by a PCR-based method for screening fosmid library (Accession No.GU723506). After the blastx comparison, the cloned gene was confirmed that it was Monascus pyrG gene with 81%sequence identity to that of Aspergillus flavus NRRL3357. It can be used as a selective marker for Monascus homologus transformation system;
2. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 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;
3. The formation of protoplast condition and the regeneration of protoplast condition of UM28 strain were optimized. Two complementation plasmids, pAOP and pMAP, were respectively transformed into protoplast of UM28 and they transformed UM28 to uridine prototrophy. Homologous transformation system using pyrG as a selection marker of UM28 is established;
4. The plasmid pGFP-pyrG, containing the Ma-pyrG gene and a GFP expression cassette, was constructed by the DNA recombinant technique. Then transform pGFP-pyrG into UM28 protoplast using pyrG as a selection marker, expression of transformed gene was evaluated using the GFP gene.
1.根据已报道的真菌乳清苷-5'-磷酸脱羧酶基因(pyrG)的氨基酸序列,采用CODEHOP策略设计简并引物,从橙色红曲菌的基因组DNA中克隆得到pyrG基因片段。然后运用PCR法筛选红曲菌Fosmid文库,获得了pyrG基因全长(GenBank登录号：CU723506)。经过blastx比较发现,红曲菌pyrG基因与Aspergillus flavus NRRL3357的氨基酸序列同源性最高,达到81%。该基因能够作为筛选标记应用于红曲菌同源转化系统；
2.以橙色红曲菌AS3.4384为出发菌株,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+220 bp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-,能够用于红曲菌同源转化系统的构建；
3.对UM28菌株的原生质体制备、再生和转化方法进行了优化,采用原生质体PEG介导转化将互补质粒pAOP和pMAP分别转入UM28中,能够使其恢复野生表型。建立了以pyrG为筛选标记的红曲菌同源转化系统；
4.通过DNA重组技术构建了表达载体pGFP-pyrG,载体中含有pyrG筛选标记和GFP蛋白表达单元。通过基因转化试验,在红曲菌中以pyrG为筛选标记进行了GFP蛋白的表达。
In this paper, pyrG gene fragment was cloned from Monascus aurantiacus genomic DNA using degenerate primers designed with CODEHOP strategy. And its complete sequence was obtained by a PCR-based method for screening fosmid library. Uridine auxotrophy strains from M. aurantiacus AS3.4384 were isolated by resistance to 5-FOA after UV mutagenesis. One positive colony was obtained and named UM28, it was further confirmed by sequencing and transformation. The formation of protoplast condition and the regeneration of protoplast condition of UM28 strain were optimized, and a homologous transformation system using pyrG as a selection marker of UM28 is established. A plasmid pGFP-pyrG containing the GFP expression cassette was constructed and transformed into UM28 protoplast, expression of transformed gene was evaluated using the GFP gene. The major findings were expounded as follows:
1. According to the known Orotidine-5'-phosphate decarboxylase, pyrG gene fragment was cloned from Monascus aurantiacus genomic DNA using degenerate primers designed with CODEHOP strategy. And its complete sequence was obtained by a PCR-based method for screening fosmid library (Accession No.GU723506). After the blastx comparison, the cloned gene was confirmed that it was Monascus pyrG gene with 81%sequence identity to that of Aspergillus flavus NRRL3357. It can be used as a selective marker for Monascus homologus transformation system;
2. A pyrG mutant strain from M. aurantiacus AS3.4384, named UM28, was isolated by resistance to 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;
3. The formation of protoplast condition and the regeneration of protoplast condition of UM28 strain were optimized. Two complementation plasmids, pAOP and pMAP, were respectively transformed into protoplast of UM28 and they transformed UM28 to uridine prototrophy. Homologous transformation system using pyrG as a selection marker of UM28 is established;
4. The plasmid pGFP-pyrG, containing the Ma-pyrG gene and a GFP expression cassette, was constructed by the DNA recombinant technique. Then transform pGFP-pyrG into UM28 protoplast using pyrG as a selection marker, expression of transformed gene was evaluated using the GFP gene.
引文
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