Construction of a cDNA Library and Cloning of an Arabinosidase cDNA from Armillariella tabescens
- VernacularTitle:假密环菌cDNA文库的构建及其阿拉伯糖苷酶基因的克隆
- Author:
Dongsheng YAO
;
Hui HUANG
;
Long ZHAO
;
Chunfang XIE
;
Daling LIU
- Publication Type:Journal Article
- Keywords:
Armillariella tabescens Bioinformatics Arabinosidase Cloning
- From:
China Biotechnology
2005;25(6):65-70
- CountryChina
- Language:Chinese
-
Abstract:
The expression cDNA library of A. tabescens was constructed by SMART technique, which useλTriplEx2 as a vector. The titer and the percentage of the constructed library were about 1.0 × 106pfu/mland 98.3% respectively, and the titer and the capacity of the amplified library were about 3.1 × 108pfu/mland 4.2 × 1010. The library was used to provide expressed sequence tags (ESTs). 147 Expressed SequenceTaqs (ESTs) were gained from 176 clones, which were selected randomly and sequenced at the 5'end. Thesequences were submitted to the EMBL database. Blasting the sequences in the GenBank, 43 of them werefound that they have significant similarity with data in GenBank. EST AJ620046 was has significantsimilarity with the arabinosidase of Bacteroides thetaiotaomicron. Using SMART-RACE a full-length cDNA ofAJ620046 was successfully obtained. In order to initially characterize the biochemical properties ofAJ620046, the ORF of AJ620046 named AF was cloned and expressed in Pichia Pastoris yeast.Recombinant pHIL-S1-AF constructed by inserting AF into pHIL-S1 was transformed into Pichia PastorisGS115. Preliminary experiments indicated that AJ620046 was expressed as a 32 kDa protein in recombinantyeast.
