Karyotype Analysis Of Penicillium Griseofulvum And Phylogenetic Reanalysis

Karyotype analysis refers to the analysis of chromosome phenotypic characteristics of cells,including the study of chromosome morphology,number,size,banding and other contents.As an important means of fungal classification and genetic research,karyotype analysis provides an important research basis and theory for the evolution and classification of fungi,chromosomal structure,morphology and variation,and gene localization.Penicillium griseofulvum belongs to Ascomycota,which can produce a variety of secondary metabolites such as griseofulvin.Up to now,the karyotype information of Penicillium griseofulvum is still blank.The determination of the chromosomal karyotype of Penicillium griseofulvum is helpful for the better development and utilization of this strain,such as the determination of the position of griseofulvin gene cluster on the chromosome,and the study of the regulation mode of related gene expression,so as to lay a foundation for the better regulation of the synthesis of griseofulvinAt present,in the identification and classification of Penicillium fungi,some names of Penicillium fungi are confused,some names are incomplete and invalid,or some names are synonyms of other species.The object of this study was Penicillium griseofulvum D-756(syn.Penicillum patulum D-756),which was obtained by Wu Songgang et al.Using Penicillum patulum 4541 as the original strain,after more than ten rounds of physical and chemical compound mutagenesis.In order to obtain the karyotype information of Penicillium griseofulvum and further clarify the taxonomic relationship of Penicillium griseofulvum.To this end,this research has carried out the following work:1.Determination of cytological karyotype of Penicillium griseofulvum 4541:By increasing the number of sections,we observed the chromosome of Penicillium griseofulvum 4541 by using the traditional and improved germ tube burst method(GTBM).The number of chromosomes of Penicillium griseofulvum 4541 was determined to be 5,and the highly concentrated chromosome morphology map of mitosis was obtained.2.Determination of cytological karyotypes of Penicillium griseofulvum D-756 and Aspergillus oryzae FS1125: The modified GTBM method was used to visualize the chromosomes of Penicillium griseofulvum D-756 and Aspergillus oryzae FS1125,and the number of chromosomes of Penicillium griseofulvum D-756 and Aspergillus oryzae FS1125 were determined to be 5 and 8 respectively,and the highly concentrated mitosis chromosome morphology maps were obtained respectively.It is also proved that the difference of chromosome number between species is obviously greater than the difference within species.The karyotypes of the mutant strain D-756 and the original strain 4541 were compared.The results showed that the number of chromosomes was the same,but there was a significant difference in the relative length of the smallest chromosome(P<0.05).3.The macroscopic morphological observation of the wild strain Penicillium griseofulvum 4541,mutant strain D-756 and wild strain 40393 showed that the original wild strain 4541 had obvious morphological changes after more than ten rounds of physicochemical mutagenesis.The spore color of the mutant strain D-756 was white.The spores of the original wild origin strain 4541 and another wild Penicillium griseofulvum40293 were grey-green in color.The gene sequences of ?-tubulin and calmodulin of wild strains 4541,mutant D-756 and wild strains 40393 were cloned and sequenced.The results showed that the ?-tubulin gene sequence of the mutant D-756 was 3?4 bases different from that of the original strain 4541 and the reference strain 40293 of Penicillium griseofulvum.The calmodulin gene sequence of the mutant D-756 was 7?8bases different from that of the original strain 4541 and the reference strain 40293 of Penicillium griseofulvum,indicating intraspecies difference.The ?-tubulin and calmodulin gene sequences of the three strains were homologous retrieved from Gen Bank database,and the NJ phylogenetic tree was constructed.Phylogenetic analysis showed that the three strains were closely related to Penicillium griseofulvum.