Construction Of CDNA Library Of Armillariella Sp.(E-20) ,Random Sequencing And Bioinformatics Analysis Of Some Clones From The Library

Aflatoxins are a group of secondary metabolites produced by different species of the genus Aspergillus, the aflatoxinBl (one of them) is the most potent toxic and carcinogenic. It is a hot topic that the bio-detoxificition of aflatoxins in the studying field about aflatoxins is. Armillariella sp.(E-20), that produce aflatoxin-detoxifizyme(ADTZ), has been screened by our research team, and from which ADTZ has been isolated. In order to make a resource information databank of the fungus, in this study, an expression cDNA library of Armillariella sp.(E-20) was constructed by SMART technique, which used A TriplEx2 as vector. The titer and the percentage of the library constructed were about 1.0 3 106pfu/ml and 98.9%; meanwhile, the titer and the capacity of the amplified library were about 3 × 108pfu/ml and 4×1010.17 Expressed Sequence Taqs(ESTs) were gained from 20 clones which were selected randomly and sequenced at 5' end. Blasting in the GenBank, 16 of them were found that they have no significant similarity with data in GenBank, which means they were new sequences, but one sample(020) of them has high homology with data of Glyceraldehyde 3-phosphate dehydrogenase ( GPD ) genes of other species in GenBank. After through-sequencing the clone and trimming the vector information, the sequence(encoded 200p) consisting 910 bp was obtained from the sequence of sample 020. It was made BlastX searched in GenBank, and the result showed the 200p was of representative of GPD in Armillariella sp.(E-20).The 200p and a amino acid sequence (encoded E20-GPD) gained from analysis of Open Read Frame(ORF) of the 200p were analyzed ulteriorly by bioinformatics methods. Analysis of ORF and coden usage indicated that there was a ORF with 639bp and coding 212 amino acids in the 200p, and a preference for particular synonymous codons. Prediction for secondary structure and function suggested that the 23rd amino acid residue-cysteine and nearby sequence (ASCTTNCL) is in a consensus pattern of Glyceraldehyde 3-phosphate dehydrogenase active site, in which the Cys is the catalytic residue, and there are some potential sites which may have biological signification in E20-GPD. Super-family analysis showed that E20-GPD has two super-families, one isGlyceraldehyde-3 -phosphate dehydrogenase-like, C-terminal domain from 23-187 amino acid residue, the other is NAD(P)-binding Rossmann-fold domain from 1-33 amino acid residue. E20-GPD was located in cytoplasm by analyzing for subcellular localization. The 3-D model was obtained by homology modeling method. A phylogenetic tree based on multi-sequences alignment of E20-GPD and other GPDs in 15 species of fungi was generated by analysis of phylogenetic. According to the results of super-family analysis of NAD(P)-binding Rossmann-fold domain, homology analysis of E20-GPD and other GPDs mentioned above and comparison between 3-D model of E20-GPD and models of templates used modeling, the whole sequence of Glyceraldehyde-3-phosphate dehydrogenase of Armillariella sp.(E-20) is 123-131 amino acids shorter than those reported in databank, and lacks a fragment of NAD(P)-binding domain of N terminal.Accomplishment of this thesis is an important initial work for building the resource information databank of Armillariella sp.(E-20).