作　　者： ; ; ; ; ;

机构地区： 华南农业大学食品学院生物工程系

出　　处： 《食用菌学报》 2012年第2期1-7,共7页

摘　　要： 利用反向长距离PCR技术从银耳(Tremellafuciformis)芽孢gpd基因出发克隆上游gpd启动子序列,将预测的gpd启动子片段与多功能纤维素基因(mfc)连接构建表达载体,与潮霉素抗性质粒pBgGl-hph共转化银耳芽孢,对拟共转化子进行酶活发酵试验。结果表明:4轮反向长距离PCR克隆得到了1761bp的上游序列,经预测启动子落在上游1000bp左右区域内,包含两个高分值的起始转录位点,将gpd启动子分成gpd-Tre1(885bp)、gpd-Tre2(708bp)、gpd-Tre3(466bp)3段区域,分别与多功能纤维素基因(mfc)构建表达载体pgTre1-mfc、pgTre2-mfc和pgTre3-mfc。拟共转化子酶活发酵试验结果发现3个表达载体的转化子均能检测到多功能纤维素酶活,转化子T1-2包含gpd-Tre1启动子大片段,整体酶活最高,CMC酶活为14.12U/mL,比出发菌株Tr01提高34.3%,比工程菌株yLes3提高25.7%,木聚糖酶活为34.8U/mL,比Tr01酶活提高26.3%,略低于yLes3。3个启动子片段均具有表达活性,相比之下885bp的大片段gpd启动子表达活性更高。 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 cellulase gene（mfc）,and co-transformed into the yeast-like conidia with the hygromycin resistance plasmid pBgGl-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.Cellulase 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.

关 键 词： 银耳 芽孢 启动子 反向长距离 多功能纤维素酶

领　　域： [农业科学] [农业科学]