Identification, Classification, And Phylogenetic Relationships Of Pathogenic Fungi Based On Mitochondrial Cytochrome B Gene And Translation Elongation Factor Gene Analysis

Fungus is a large kind of organism, which exists in nature broadly. The hyphomycete such as Aspergillus, Fusarium, Paecilomyces and so on has a widely application in numerous areas, such as industry, agriculture, medicine, environment etc. they may induce opportunitic infection and intoxication by fungus toxin in patients with immune deficiency. Some may be involve in the development and progression of malignant tumor; some is likely cause milden and rot of foodstuff and forage. The irritant, organic acid, organized enzyme produces by fungi can be use for industry and agriculture such as brewage. Some may play an active role as it has been used in biocontrol against insects. It is of significance to the classification, identification and phylogeny of the strains.Comparing with nuclear DNA, mitochondrial cytochrome b gene possesses several features, for instance, maternal inheritance, simple structure, convenient manipulation, high sensitivity and so on. Owning to comparatively large variation in interspecies, and certain conservatism in the intraspecies, it is extremely easy to discovery the diversity of close relationship species. So mitochondrial cytochrome b gene is deemed to be an ideal molecular marker in the aspects of evolution and phylogeny. Recently it is applied in many species, and gained an attention in fungal identification and classification.Translation elongation factor gene (EF1A), because of high genetic conservatism, provides signification value which studies genetic relationship. As a molecular marker, translation elongation factor gene (EF1A) is used for studying in fungal identification and classification increasingly. Through analyzing the data base of DNA or amino acid sequence, then evaluating and consummating the evolution relationship of fungus, translation elongation factor gene (EF1A) will play a gigantic economical effect in agriculture and medical science.The fungi isolated from clinic and environment was identificated by morphology , in preliminary. The strains from clinic and that with difficulties or mistakes in identification by morphology has been identified and classified by the approach of mitochondrial cytochrome b gene analysis.Retrieving in GenBank at first, design the fungi translation elongation factor consensus primer and check the its versatility . According to the DNA and amino acid sequence obtained study the classification and phylogeny among the fungi strains.Depend on analyzing cytb gene sequences from fungi that Aspergillus (26 strains), Fusarium (30 strains), Paecilomyces (22 strains), Acremonium (12 strains) which are clinical isolated and have difficulties in identification by morphology. Some strains has been reidentefied and reclassified with morphology characteristics. There are 20 strains of Aspergillus (17 Aspergillus fumigatus, 2 Aspergillus terreus, 1 Aspergillus flavus), 21 strains of Fusarium (17 Fusarium solani, 1 Fusarium moniliforme, 1 Fusarium subglutinans, 2 Fusarium verticillioides) and 11 Penicillium(6 Penicillium chrysogenum, 1 Penicillium aurantigriseum, 1 Penicillium crustosum, 2 Penicillium oxalicum,1 Penicillium expansum)has been identified.The fungi universal primer has been designed by comparing translation elongation factor sequence of 18 strains fungi from GenBank. The fungi include Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Cryptococcus neoformans, Coccidioides immitis, Mucor and so on. The 750-850bp specific string was obtained by PCR amplification of 15 genus, 40 species, 120 strains fungi which are consisted of Aspergillus, Fusarium, candida, Paecilomyces, Penicillium, Mucor and so on.40 strains of Paecilomyces'TEF gene sequences were gained , of which the lengths are all 745bp. And the TEF amino acid sequence presumed is 248aa based on the Yeast Mitochondrial Code . To contribute phylogenetic tree ,research and ensure the system classification of each strain.It is a new field that the study the classification, identification and phylogeny of fungi by mitochondrial cytochrome b gene which has been well-accepted . At present, the report is seldom.The research has analyzed the identification and classification of major pathogenitic fungi isolated from clinic and fungi with difficulties in identification by morphology. The report involved hasn't been found.The fungi universal primer has been designed by comparing translation elongation from GenBank. And it is verificated in 15 genus fungi which contain Aspergillus, Paecilomyces, Fusarium and so on. It is the first try to study the classification and phylogenetic relationship of Paecilomyces with TEF gene sequence analysis which never reported. The analysis of mitochondrial cytochrome b gene and translation elongation factor gene from fungi has the extremely advantage in identifying fungi exactly especially in the isolates with difficulties in identification. Moreover, it is highly demonstrated in the research of classification and phylogenetic relationship of fungi. The design of fungi universal primer of TEF gene has enriched and consummated the research of classification, identification and phylogenetic relationship in fungi. The study has provided data and scientific evidence for classification, evolution, ecology distribution, diagnosis,treatment in mycosis and epidemiology in fungi.