【作者】 陈金卿；

【导师】 黄建忠；

【作者基本信息】 福建师范大学 ， 微生物学， 2008， 硕士

【摘要】 为验证海洋破囊壶菌△~4-脱饱和酶cDNA序列的基因功能,构建重组表达质粒,并以酿酒酵母作为宿主进行功能表达分析,添加外源脂肪酸C22:5底物,半乳糖诱导表达后,阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪酸含量的41.13%),△~4-脱饱和酶基因(FAD4)在酿酒酵母中得到了表达。利用重叠延伸PCR技术扩增△~5-延长酶基因(ELO5)和FAD4的融合基因ELO5-FAD4,测序验证后构建重组表达质粒,转化酿酒酵母并添加外源脂肪酸C20:5底物,半乳糖诱导表达。与对照菌相比,阳性克隆子总脂肪酸中出现了与二十二碳六烯酸C22:6甲酯标准品相对应的新峰,从而获得更经济的DHA工程菌株。为进一步了解FAD4转录调控的分子机制,通过LA-PCR方法,从破囊壶菌基因组DNA中扩增得到约1000bp的FAD4 5’端侧翼区域的单一产物,并进行序列测定及提交GenBank数据库(GenBank accession No.EU074209)。经启动子软件与序列比对分析,该序列分别有类似于TATA盒、CAAT盒、GC盒等元件,这些元件均是真核启动子序列的基本结构特征。对FAD4 5’端侧翼区域进行片段缺失扩增,得到系列亚克隆片段,连接到以绿色荧光蛋白(GFP)为报告基因的pGlow-TOPO质粒中,构建了系列重组表达质粒,并成功转化大肠杆菌E.coli TOP10。荧光显微观察表明阳性转化子均发出绿色荧光,它们启动绿色荧光蛋白表达的功能得到确认。更多还原

【Abstract】 In order to determine the function ofΔ~4-desaturase of Thraustochytrium sp.FJN-10 (FAD4),the recombinant plasmid was constructed and transformed into Saccharomyces cerevisiae strain INVScl for expression.It was found to exhibitΔ~4-fatty acid desaturase activity in the presence of exogenous fatty acid substrate docosapentaenoic acid(100μmol/L)under introduction of GAL1 and DHA reached 41.13%of the total yeast fatty acid by GC detection.It was suggested that the protein encoded by FAD4 could specifically catalyze DPA into DHA.The fusion gene ofΔ~5-elongase gene(ELO5)and FAD4 was amplified by overlap extension PCR and sequenced.The recombinant plasmid containing target gene was constructed and transformed into Saccharomyces cerevisiae for expression by supplemented with 0.3 mmol/L Eicosapentaenoic acid.A novel peak corresponding to DHA methyl ester standard was detected with the same retention time which was absent in the cell transformed with empty vector.So to obtain the much more economical engineering strain which could produce DHA.A 5’ flanking region DNA encoding FAD4 with about 1000bp was specifically amplified by LA-PCR,then sequenced and submitted into the GenBank data(GenBank accession No.EU074209).The sequence contained a novel promoter region with TATA box、CAAT box and GC box-like elements in the identical positions shared by the eucaryote promoter sequence regions in comparison with the sequences in the GenBank.The amplified sub-cloned products of 5’ flanking region were ligated into the vector pGlow-TOPO which contained the green fluorescent protein(GFP)gene as the reporter gene to construct the expression plasmids.The recombinant plasmids were transformed into Escherichia.coli TOP10,respectively.Many Escherichia.coli cells with the recombinant plasmid could emit fluorescence.The result indicated that the 5’ flanking region had the promoter gene segment,which could promote GFP gene to express in Escherichia.coli cells.更多还原