Development Of A Homologous Transformation System For Monascus Aurantiacus Based On The PyrG Gene

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.