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从球等鞭金藻、眼虫、高山被孢霉和拟南芥中分别克隆到Δ9链延长酶、Δ8去饱和酶、Δ5去饱和酶和Δ15去饱和酶基因,利用我们的多基因聚合方法,将这4个基因聚合到植物表达载体pCambia2300上,其中每个基因都含有独立的CaMV35S启动子和Tnos终止子。利用农杆菌介导法将该表达载体转入棉花,通过卡纳霉素和PCR筛选获得转基因阳性植株,提取转基因阳性植株叶片总脂肪酸,用气相色谱分析法检测到花生四烯酸(AA,20:4Δ5,8,11,14)和二十碳五烯酸(EPA,20:5Δ5,8,11,14,17)含量分别达1.0%和5.0%。表明通过基因代谢工程在棉花中异源合成EPA是可行的,为进一步在棉籽中生产VLCPUFAs奠定了基础。
The Δ9 chain elongase, Δ8 desaturase, Δ5 desaturase and Δ15 desaturase genes were cloned from Isochrysis galbana, Euglena, Mortierella alpina and Arabidopsis thaliana, respectively, using our polygene method These four genes were polymerized into the plant expression vector pCambia2300, each of which contained the independent CaMV35S promoter and Tnos terminator. The expression vector was transferred into cotton using Agrobacterium tumefaciens method. The positive transgenic plants were obtained by screening of kanamycin and PCR. The total fatty acids in transgenic plants were extracted. The contents of arachidonic acid (AA, 20 : 4Δ5,8,11,14) and eicosapentaenoic acid (EPA, 20: 5Δ5,8,11,14,17) content of 1.0% and 5.0% respectively. This indicates that it is feasible to heterosecondly synthesize EPA in cotton through gene metabolic engineering and lay the foundation for further production of VLCPUFAs in cottonseed.