【作者】 夏晓峰；

【导师】 黄建忠； 江贤章；

【作者基本信息】 福建师范大学 ， 发酵工程， 2009， 硕士

【摘要】 破囊壶菌Thraustochytrium能够合成大量高度不饱和脂肪酸（PUFA）,为探究海洋真菌Thraustochytrium PUFA的生物合成途径,本课题从Thraustochytriumsp.FJN-10中克隆到了PUFA合成相关的Δ5-脂肪酸脱饱和酶基因的cDNA序列（GenBank Accession Number:EU643618）,该序列全长1320bp,编码439个氨基酸,分子量为49.82 KDa,等电点为8.71。该蛋白质序列含有三个保守的组氨酸富集区,且第三个组氨酸保守盒的组氨酸被替换成谷氨酰胺,N-端含有类细胞色素-b5结构域,具有脱饱和酶的典型一级结构特征;氨基酸序列比对分析表明该序列与破囊壶菌Thraustochytrium sp.ATCC21685、Pavlova salina、Perkinsus marinus的Δ5-脂肪酸脱饱和酶分别具有99%、49%和47%的一致性,此外该序列还分别与Mantoniellasquamata和Ostreococcus tauri的Δ6-脂肪酸脱饱和酶具有42%和41%的一致性;进化树分析表明该酶属于Δ5-脂肪酸脱饱和酶家族。将该序列与酵母表达载体pYES2连接构建重组表达载体pYFAD5,并转入酿酒酵母构建重组工程菌,在含底物二高-γ-亚麻酸(di-hono-γ-linoleic acid,DGLA,20:3Δ^8,11,14n6)的培养基中添加半乳糖诱导表达,提取总脂肪酸,气相色谱-质谱分析表明重组工程菌能将底物DGLA转化成花生四烯酸(Arachidonic acid,AA,20:4Δ^5,8,11,14n6),转化率为56.40%。上述结果表明从海洋破囊壶菌Thraustochytrium sp.FJN-10中克隆的脂肪酸脱饱和酶基因的编码产物具备Δ5-脱饱和酶的功能。利用LA-PCR法克隆得到了海洋破囊壶菌Thraustochytrium sp.FJN-10Δ5-脱饱和酶基因的5′端侧翼序列（GenBank Accession Number:EU918393）,利用多种在线软件对该序列进行分析,分析结果表明该序列包含了多种启动子的特征元件。将该序列与人、斑马鱼、恒河猴、线虫以及褐鼠的Δ5-脱饱和酶基因5′端侧翼序列进行多序列比对,采用邻位相连算法构建距离进化树,进化树结果表明破囊壶菌Thraustochytrium sp.FJN-10的Δ5-脱饱和酶基因的5’端侧翼序列与人类和恒河猴进化距离最近,破囊壶菌Thraustochytrium sp.FJN-10的Δ5-脱饱和酶基因的调控机制比较接近高等脊椎动物,此外FootPrinter的分析也支持了上述结果。为了验证海洋破囊壶菌Δ5-脂肪酸脱饱和酶基因5’端侧翼序列的启动子功能,将该5’端侧翼序列克隆到真核启动子验证表达载体pYGFP质粒中的绿色荧光蛋白基因上游,以真核生物酿酒酵母作为转化的宿主菌,利用荧光显微镜观察酵母的荧光激发状态,实验结果表明来自于破囊壶菌Δ5-脂肪酸脱饱和酶基因5’端侧翼序列能够在酵母细胞中启动GFP基因的表达,使酵母细胞在荧光显微镜的激发下产生绿色荧光,表明克隆的破囊壶菌Δ5-脂肪酸脱饱和酶基因5’端侧翼序列具有启动子的功能。更多还原

【Abstract】 A cDNA with homology to fatty acid desaturases was selected fromThraustochytrium sp.FJN-10 (GenBank Accession Number: EU643618) , which containsan N-terminal cytochrome b5-like domain as well as three histidine-rich domains that is acommon feature of primary structure of fatty acid desaturase, the third histidine boxcontains a histidine to glutamine substitution(HXXHH--QXXHH). The coloned ORF was1320bp encodes a protein with 439 amino acids, its molecular weight is 49.82 KDa, andthe pI is 8.71. Homology analysis suggest the sequence was 99% identities with theΔ5-desaturase of Thraustochytrium sp. ATCC21685 and 49%, 47% with theΔ5-desaturase of Pavlova salina, Perkinsus marinus respectively, the sequence alsoshare 42%, 41% identities with theΔ6-desaturase of Mantoniella squamata andOstreococcus tauri respectively. Its desaturase activity was confirmed by expression of itunder the inducible promoter of galactose in Saccharamyces Cerevisiae in the present ofDGLA as substrate, total lipids were extrated from them, a new fatty acid was detected inthe lipid fraction by GC-MS (gas chromatography-mass spectrum), the results suggestedthe recombinant cell converted the DGLA to AA, with a conversion efficiency of 56.40%.Therefore, the cDNA cloned from Thraustochytrium sp.FJN-10 expressed in S. cerevisiaecan introduce aΔ5 double bond into DGLA, converted DGLA to AA, this result indicateditsΔ5-desaturase activity in S. cerevisiae.The 5’ flanking region ofΔ5-desaturase gene was cloned by LA-PCR and sequenced (GenBank Accession Number: EU918393), then it was analyzed by many on-line softs which all suggested the region contains many significant characteristics of promoter. Characterization and comparison of this sequence with the 5’ flanking region ofΔ5-desaturase gene from Human, Zebrafish, Rhesus, C.elegan, Rattus norvegicus, phylogenetic tree was constructed by Neighbor-Joining method , the result showed that Thraustochytrium sp.FJN-10 share the smallest evolution distance with Human and Rhesus which implied Thraustochytrium sp.FJN-10 may share the same regulation mechanism of the expression ofΔ5-desaturase gene with higher vertebrates, this conclusion was supported by FootPrinter analysis again. In order to verified the promoter function of the 5’ flanking region ofΔ5-desaturase gene, we have constructed a promoter function identification vector pYGFP, ligated this sequence with pYGFP vector which located at the upstream of GFP to constructed recombinant plasmid pYD5GFP, then pYD5GFP was transformed into Saccharamyces Cerevisiae. The GFP(green fluorescent protein) gene was expressed in Saccharamyces Cerevisiae under the regulation of this sequence, fluorescence was assayed by microscope. The result suggested that the recombinant cells yielded a green fluorescent when they were excited by the corresponding wavelength light which indicated its full function of promoter.更多还原