Mucor sp.EIM-10 C18-Δ^(15)脂肪酸脱氢酶启动子区域的克隆和功能鉴定  

Cloning and Function Identification of the Promotor of Δ^(15)-Fatty Acid Desaturase Gene from Mucor sp.EIM-10

作　　者：毛若雨[1] 江贤章[1] 陈晓峰[1] 田宝玉[1] 刘静[1] 吴松刚[1] 黄建忠[1] 

机构地区：[1]福建师范大学工业微生物教育部工程研究中心生命科学学院,福建现代发酵技术工程研究中心,福建福州350108

出　　处：《药物生物技术》2010年第1期15-20,共6页Pharmaceutical Biotechnology

基　　金：国家自然科学基金(30970047;30370028);福建省自然科学基金重大项目(2003F005);福建省自然科学基金(2008F3036);福建省发改委科技产业化项目([2005]847);福建省科技平台(2005Q007)

摘　　要：应用LA-PCR技术,扩增得到1291bp的Mucor sp.EIM-10Δ15脂肪酸脱氢酶启动子区域的单一产物。对该产物测序,经序列比对分析,发现该序列具有真核启动子序列的基本结构特征,含有TATA盒、CAAT盒、GC盒等元件。根据本中心已构建的质粒PYD15PMCD15和质粒PYGFP,构建启动子功能鉴定阴性质粒PYMCD15,将扩增得到的Δ15脂肪酸脱氢酶启动子区域序列连接到PYMCD15中,构建重组表达质粒PYMD15PMCD15,转化Saccharomyces cerevisiae尿嘧啶营养缺陷型INVSc1。发酵诱导后GC/MS分析,结果表明Δ15脂肪酸脱氢酶启动子区域能够启动Mucor sp.EIM-10Δ15脂肪酸脱氢酶基因的表达。A 5＇ flanking region DNA of △^15- fatty acid desaturase from Mucor sp. EIM- 10 was specially ampli- fied by LA PCR, which was cloned into pMD- 18 T vector and sequenced. The sequence contained a novel promoter region with TATA box, CAAT box and GC-box like elements in the identical positions common to the eukaryote promoter sequence regions. According to the plasmid of PYD15PMCD15 and PYGFP, the plasmid of PYMCD15 was constructed. The amplified product of promotor region was ligated into the PYMCD15 vector which contained the △^15- fatty acid desaturase gene to construct the expression plasmid PYMD15PMCD15. The recombined plasmid was transferred into Saccharomyces cerevisiae strain INVScl. The GC/MS analysis on the profile of the total fatty acid demonstrated that the novel peak for product ALA appeared in the transgenetic yeast compared with the control yeast containing the original vector PYMCD15.

关 键 词：MUCOR sp.EIM-10 脂肪酸脱氢酶 LA PCR 启动子 

分 类 号：Q55[生物学—生物化学]