| Aspergillus has a close relationship with human daily life. It is a saprophytic fungus that plays an essential role in recycling environmental carbon and nitrogen, it also played a central role in the production of traditional fermented foods such as soy sauce, and soybean paste and rice wine throughout the last thousand years. Recently more than 1200 biological metabolites have been discovered in metabolites of aspergillus strains. On the other hand, many fungal toxins called mycotoxins are also producted by species of aspergillus. For example, aflatoxins, which are capable of causing disease in humans and other animals. Some aspergillus species such as A.fumigatus, A.flavus, A.terreus and A.niger are commonly associated with invasive infection in humans. There have been reports suggesting that the causative agents of aspergillus are species specific. Over the past 10 years, A. fumigatus has become the most prevalent airborne fungal pathogen, causing severe and usually fatal invasive infections in immunocompromised hosts in developed countries. Invasive aspergillosis (IA) has been observed was responsible for approximately 30% of fungal infections in patients dying of cancer, and it is estimated that IA occurs in 10% to 25% of all leukemia patients, IA is now a major cause of death at leukemia treatment centers and bone marrow transplantation (BMT) and solid-organ transplantationunits.The aim of this study is to examine aspergillus strains as to their phylogenetic relationships. Pylogenetic analysis was based on DNA sequences of theβ-tubulin gene, GAPDH gene, MTO1 gene and HSP70 gene partial encoding regions and Multi-gene phylogenetic analyses were also conduct to address the evolution of the 26 aspergillus species. Separate analyses and the combined analyses of the four genes were conducted. The base frequeney, base substitution and genetic distance (p-dictance) of DNA sequence have been analyzed by using two biosoftwares ClustalX 2.0 and MEGA 4.1. Pylogenetic tree of the 26 aspergillus was constructed used NJ (neighbor joining) method. Data and result are presented here from portions sequences of four loci and the combined datas. Summarizing the outcomes, we can conclude the results As follows:To theβ-tubulin gene of the 26 aspergillus species, the PCR amplification fragments ,there are seven aspergillus species haven't intron sequences, they are A. wentii,A. tamarii,A. subolivaceus,A. parasiticus,A. oryzae,A. flavus,A. cristatus. The other 19 aspergillus species all have introns, which range from 48 bp to 84bp. The extron sequences are 456 bp and the translated protein contained 152 amino acids. To the GAPDH gene of the 26 aspergillus species, PCR amplification fragments of the A. terrus,A. terricola,A.glaucus,A. clavatus species have not intron; the other 22 aspergillus species have intron, which range from 53 bp to 60 bp. The extron sequence of the eight aspergillus species (A.tamarii,A.wentii,A.subolivaceus,A.oryzae,A.fumigatus,A.flavus,A.brunneo-uniseriatus,A.parasiticus) is 555 bp, the protein of these eight aspergillus species have 185 amino acids. The other aspergillus species extron is 552 bp, which protein contains 184 amino acids. The MTO1 gene of the 26 aspergillus species haven't introns, But A.nidulans and A.aculeatus extron are 515 bp, the others strains extron are 512bp. The entire HSP70 genes haven't intron sequences, the extron sequences are 439 bp. The protein sequences of HSP70 gene have 146 amino acids.The A+T avergage content of theβ-tubulin gene is 41.4% and G+C content is 58.6%, indicated theβ-tubulin gene is G+C bias. The GAPDH gene A+T content is 42%, G+C content is 58%, the G+C content more than A+T content, shows the GAPDH gene is G+C bias. The MTO1 gene A+T content is 45.1%, G+C content is 54.8%, the G+C content more than A+T content. The A+T contant of HSP70 gene is 44.3%, G+C content is 55.7%, the G+C content more than A+T content, also shows the HSP70 gene is G+C bias.β-tubulin gene of the 26 aspergillus species sequences have 428 identical pairs, have 22 transitionsal pairs and 4 transversional pairs. The average transition and transversion ratio ofβ-tubulin gene sequence is 5.5; GAPDH gene of the 26 aspergillus species sequences have 439 identical pairs, have 34 transitionsal pairs and 35 transversional pairs. The average transition and transversion ratio ofβ-tubulin gene sequence is 0.97; MTO1 gene of the 26 aspergillus species sequences have 421 identical pairs, have 41 transitionsal pairs and 42 transversional pairs. The average transition and transversion ratio ofβ-tubulin gene sequence is 0.98; HSP70 gene of the 26 aspergillus species sequences have 401 identical pairs, have 26 transitionsal pairs and 9 transversional pairs. The average transition and transversion ratio ofβ-tubulin gene sequence is 2. 89;The average P-distance ofβ-tubulin gene is 0.06, Most of them vary from 0 to 0.11; the average p-distnace of GAPDH gene is 0.11, Most of them vary between 0 and 0.19; the average p-distnace of MTO1 gene is 0.18, Most of them vary between 0 and 0.14; the average p-distnace of HSP70 gene is 0.08, Most of them vary between 0 and 0.14.The result shows the MTO1gene has more evolution rates than the other three genes.Theβ-tubulin gene, GAPDH gene, HSP70 gene and MTO1 gene combined sequences datas were also analysised. The total sequences of the combined genes range from 1959 to 1962 base pairs. Among them the conserved sites numbers is 1318, the variable sites are 647. The average A+T content is 43.2%, G+C average content is 56.8%. For the combined datas the identical pairs is 1745 base pairs, transitionsal pairs is 130 and transversional pairs is 85. The average transition and transversion ratio of combined gene datas sequence is 1.52. The transitionsal pairs are more than transversional pairs, the transitionsal pairs mainly on the second site of the codon. The transitionsal pairs is mainly T-C, transversional pairs is mainly C-G. The combined datas p-distance vary from 0 to 0.17, the average p-diatance is 0.11.The phylogentic tree of the four genes and the combined datas construct by NJ method. Among the five pylogenetic trees, the combined tree has the well-supported rates. The phylogenetic relationship within the 26 aspergillus species shows as follow:The total 26 aspergillus species is divided into six clusters.A.parasiticus, A.subolivaceus, A.wentii, A.flavus, A.oryzae, A.tamarii, A.cristatus and A.glaucus aspergillus species have the closer relationships in pylogenetic tree. In morphology classification, most of this aspergillus belongs to Flavi Section. A.brunneo-uniseriatu, A.fumigatus and A.clavatus have a close relationship. In fact A.terrus and A.terricola are the same species. A.nidulans and A.versicolor have a close relationship. A.foetidus and A.tubingensis in the same clade; A.awamori, A.niger, A.flavo-furcatis, A.candidus, A.phoenicis, A.japonicus and A.usamii in the same clades; A.carbonarius in the other clade. In morphology classification, most of this aspergillus belongs to section Nigri. A real-time PCR method for the detection of A. fumigates, A.niger and A.nidulans were established. The specific primers and Taqman probes were derived from Mto1 gene (A. fumigatus) and GAPDH gene (A.niger, A.nidulans) respectively. 26 species and some others pathogen fungi strains were used, No cross-amplification was observed. The lowest detection limits of the real-time PCR was 1.07×10-9μg/ml of the A. fumigates DNA template, 4.03×10-12μg/ml of the A.nidulans DNA template and 2.78×10-10μg/ml A.niger DNA template. Real-time PCR is a time-efficient, specifically, effective and easy-to-use method, it is a novel tool for identifying and determining the strain of pathogenic aspergillus. This method may be useful for epidemiological studies and surveillance of A. fumigates A.nidulans and A.niger in clinical samples.
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