CAJ | 학술논문

二十二碳六烯酸(Docosahexaenoic acid,DHA C22:6n-3)是具有各种重要生理功能的高度不饱和脂肪酸.分别以质粒pYTFD5和pYFAD4为模板,扩增获得860 bp的△5-延长酶基因(elo5)和1 600 bp的△4-脱饱和酶基因(fad4).利用重叠延伸PCR构建elo5-fad4融合基因,Hind Ⅲ/Sph Ⅰ双酶切后连接到经同样处理过的pYES2.0载体,构建重组表达质粒pYEL05-FAD4.转化酿酒酵母尿嘧啶缺陷型菌株INVScl,通过缺少尿嘧啶的选择性培养基筛选阳性克隆子.添加外源脂肪酸C20:5底物.半乳糖诱导表达.气相色谱-质谱分析表明,重组酵母总脂肪酸中出现了DHA(二十二碳六烯酸,C22:6n-63)新产物,融合基因elo5-fad4在酿酒酵母中得到了表达.图7表1参18
이십이탄륙희산(Docosahexaenoic acid,DHA C22:6n-3)시구유각충중요생리공능적고도불포화지방산.분별이질립pYTFD5화pYFAD4위모판,확증획득860 bp적△5-연장매기인(elo5)화1 600 bp적△4-탈포화매기인(fad4).이용중첩연신PCR구건elo5-fad4융합기인,Hind Ⅲ/Sph Ⅰ쌍매절후련접도경동양처리과적pYES2.0재체,구건중조표체질립pYEL05-FAD4.전화양주효모뇨밀정결함형균주INVScl,통과결소뇨밀정적선택성배양기사선양성극륭자.첨가외원지방산C20:5저물.반유당유도표체.기상색보-질보분석표명,중조효모총지방산중출현료DHA(이십이탄륙희산,C22:6n-63)신산물,융합기인elo5-fad4재양주효모중득도료표체.도7표1삼18
Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has many positive effects on diseases. Δ~5-elongase gene (elo5, 860 bp) and Δ~4-desaturase gene (fad4, 1 600 bp) were amplified by PCR using plasmid pYTFD5 and pYFAD4 as templates, respectively. Elo5-fad4 fusion gene amplified by overlap extension PCR was digested by Hind III and Sph I and subcloned into the yenst-Escherichia coli shuttle vector pYES2.0. The recombinant plasmid pYELO5-FAD4 containing target gene was transformed into Saccharomyces cerevisiae strain INVScl and the recombinant yeast cells were selected on agar synthetic medium lacking uracil. Expression of the fusion gene in transformant was induced by the addition of galactose to 2% (w/V). The yeast culture medium was supplemented with exogenous fatty acid substrate, eicosapentaenoic acid. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters were analyzed by GC detection. A novel peak corresponding to DHA (docosahexaenoic acid, C22:6n-3) methyl ester standards which was absent in the cell transformed with empty vector was detected with the same retention time and mass analysis. These results indicated that the protein encoded by elo5-fad4 could specifically catalyze EPA to DHA. Fig 7, Tab 1, Ref 18