超长链多不饱和脂肪酸在棉花中的异源合成

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。

Production of Very Long Chain Polyunsaturated Fatty Acids in Cotton

We have isolated four genes encoding a Δ9 elongase, a Δ8 desaturase, a Δ5 desaturase, and a Δ15 desaturase from Isochrysis galbana, Euglena gracilis, Mortierella alpina and Arabidopsis thaliana respectively. Using a multigene transfer technology that we developed, these genes were stacked together in the plant expression vector pCambia2300. Each gene contained its own CaMV35S promoter and Tnos terminator.This plant expression vector was then transferred into cotton by Agrobacterium-mediated transformation method. Transgenic cotton seedlings were first identified by screening them based on kanamycin-containing media and followed by PCR with gene-specific primers of the four transgenes. Finally, these transgenic plants were subjected to gas liquid chromatography analysis for their fatty acid composition and the results showed that the contents of arachidonic acid (ARA, 20:4Δ^5,8,11,14) and eicosapentaenoic acid (EPA, 20:5Δ^5,8,11,14,17) were 1.0% and 5.0% respectively in the leaves of the transgenic plants, indicating that the four genes were expressed incotton. Therefore, our data clearly demonstrated the feasibility for the heterologous production of EPA in cotton and this will lay a foundation for the production of VLCPUFAs, including EPA and DHA in cotton seed through transgenic technology in the future.