Random Sequencing Of Armillariella Tabescens CDNA Library And Cloning,Expression,Bioinformatics Analysis Of A Full-length CDNA For Arabinosidase

Armillariella tabscens is a fungus of Tficholomataceae, it is parasitism on the body or root of tree, and can be cause many tree's decayed diseases. According to the literature , A. tabescens is a non-toxic, edible fungus with curative properties in the treatment of cholecystitis, hepatitis, appendicitis and otitis media, and some of the fungus can decompose mycotoxins.In order to identified the potential target of A. tabescens, the expression cDNA library of A. tabescens were constructed by SMART technique which used ATriplEx2 as vector. The library was used to provide expressed sequence tags (ESTs). Large-scale sequencing of clones from cDNA library is a rapid and efficient way of discovering novel genes expressed in A. tabescens. In this paper, 120 Expressed Sequence Taqs (ESTs) were gained from 150 clones which were selected randomly and sequenced at the 5' end. The sequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 41 of them were found to have significant similarity with data sequences in the GenBank. EST AJ620046 has significant similarity with arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE we successfully obtained a full length cDNA of AJ620046. Super-family analysis showed that AJ620046 belongs to alpha-L-arabinanase, a member of arabinosidase family of enzymes. In order to initially characterize the biochemical properties of AJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast. Recombinant pHIL-S₁-AF was constructed by inserting AF into pHIL-S1 and linearized with Sal I to transform into Pichia Pastoris GS115.The recombinant stain whose phenotype was Mut⁺HIS⁺ was screened by MM and MD media. Preliminary experimentsindicated that AJ620046 was expressed as a 32 kDa protein in recombinant yeast. This is the first description of a cDNA library from A. tabscence and the presence of novel sequences within this library makes it a valuable and unique resource for studying gene expression and regulatory mechanisms that underlie the parasitism of A. tabscence. This work provided important basic research for further exploitation of A. tabscence.