Isolation and Functional Analysis of a Gpd-Tf Promoter from Yeast-like Conidia of Tremella fuciformis
A 1761 bp upper flanking sequence of the gpd promoter was isolated from yeast-like conidia of Tremella fuciformis by a four-step amplification using long distance inverse PCR (LD-IPCR). After sequence analysis, expression vectors were constructed with the gpd promoter and a multi-function cellulasc gene (mfc), and co-transformed into the yeast-like conidia with the hygromycin resistance plasmid pBgG1-hph. This sequence contained the gpd promoter, which was located in the upper 1000 bp, together with two high score transcription initiation sites. The gpd promoter sequence was divided into three functional sections, designated gpd-Tre1 (885 bp), gpd-Tre2 (708 bp) and gpd-Tre3 (466 bp), which were used to construct the expression vectors pgTre1-mfc, pgTre2-mfc and pgTre3-mfc, respectively.Cellulasc and xylanase activity was detected in submerged cultures of three putative transformants. Highest CMCase levels (14. 12 U/mL) were produced by the transformant T1-2 containing the gpd-Tre1 promoter sequence, which were 34. 3% and 25.7% higher compared with the control strain Tr01 and an engineered strain yLes3, respectively. T1-2 also exhibited the highest xylanase activity (34.8 U/mL), which was 26.3% higher compared to Tr01 but slightly lower than yLes3. Our data indicated all three parts of the gpd promoter sequence had expression activity, with the highest activity associated with the gpd-Tre1 885 bp segment.
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万方数据
维普
