銀耳芽孢內(nèi)源gpd啟動(dòng)子的克隆與功能鑒定
Isolation and Functional Analysis of a Gpd-Tf Promoter from Yeast-like Conidia of Tremella fuciformis
【作者】 聶燕華； 林俊芳； 王杰； 尤琳烽； 郭麗瓊；
【Author】 NIE Yanhua1,2,LIN Junfang1,3,WANG Jie1,3,YOU Linfeng1,3,GUO Liqiong1,31College of Food Sciences,South China Agricultural University,Guangzhou,Guangdong 510640,China;2Department of Tropical Crops,Guangdong AIB Polytechnic University,Guangzhou,Guangdong 510507,China; 3Institute of Biomass Research,South China Agricultural University,Guangzhou,Guangdong 510640,China
【機(jī)構(gòu)】 華南農(nóng)業(yè)大學(xué)食品學(xué)院生物工程系； 廣東農(nóng)工商職業(yè)技術(shù)學(xué)院熱作系； 華南農(nóng)業(yè)大學(xué)生物質(zhì)能研究所；
【摘要】 利用反向長(zhǎng)距離PCR技術(shù)從銀耳(Tremellafuciformis)芽孢gpd基因出發(fā)克隆上游gpd啟動(dòng)子序列,將預(yù)測(cè)的gpd啟動(dòng)子片段與多功能纖維素基因(mfc)連接構(gòu)建表達(dá)載體,與潮霉素抗性質(zhì)粒pBgGl-hph共轉(zhuǎn)化銀耳芽孢,對(duì)擬共轉(zhuǎn)化子進(jìn)行酶活發(fā)酵試驗(yàn)。結(jié)果表明:4輪反向長(zhǎng)距離PCR克隆得到了1761bp的上游序列,經(jīng)預(yù)測(cè)啟動(dòng)子落在上游1000bp左右區(qū)域內(nèi),包含兩個(gè)高分值的起始轉(zhuǎn)錄位點(diǎn),將gpd啟動(dòng)子分成gpd-Tre1(885bp)、gpd-Tre2(708bp)、gpd-Tre3(466bp)3段區(qū)域,分別與多功能纖維素基因(mfc)構(gòu)建表達(dá)載體pgTre1-mfc、pgTre2-mfc和pgTre3-mfc。擬共轉(zhuǎn)化子酶活發(fā)酵試驗(yàn)結(jié)果發(fā)現(xiàn)3個(gè)表達(dá)載體的轉(zhuǎn)化子均能檢測(cè)到多功能纖維素酶活,轉(zhuǎn)化子T1-2包含gpd-Tre1啟動(dòng)子大片段,整體酶活最高,CMC酶活為14.12U/mL,比出發(fā)菌株Tr01提高34.3%,比工程菌株yLes3提高25.7%,木聚糖酶活為34.8U/mL,比Tr01酶活提高26.3%,略低于yLes3。3個(gè)啟動(dòng)子片段均具有表達(dá)活性,相比之下885b...
【Abstract】 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...
【關(guān)鍵詞】 銀耳； 芽孢； gpd啟動(dòng)子； 反向長(zhǎng)距離PCR； 多功能纖維素酶；
【Key words】 Tremella fuciformis； yeast-like conidia； gpd promoter； long distance inverse PCR； multi-functional cellulase；
【基金】 國(guó)家高技術(shù)研究發(fā)展計(jì)劃(編號(hào):2006AA10Z301);國(guó)家自然科學(xué)基金(編號(hào):30371000,30671457)的部分研究?jī)?nèi)容
【分類號(hào)】S567.34