EPA和DHA合成表达载体构建及对水稻的遗传转化

The Construction and Transformation of Expression Vector into Rice for Synthesis of EPA and DHA

二十碳五烯酸(eicosapentaenoic acid, EPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)是最具有营养价值的ω-3超长链多不饱和脂肪酸(very long-chain polyunsaturated fatty acids, VLPUFAs),其主要来源是某些深海鱼油、藻类和微生物。由于过度捕捞、重金属污染及提取产生的高昂价格等问题,已难以满足市场的需求,而高等植物中不存在ω-3 VLPUFAs途径。因此,开发富含EPA和DHA新植物资源具有广阔的应用前景。本研究将7个与ω-3 VLPUFAs合成相关的酶基因共同导入水稻中,创建ω-3 VLPUFAs合成通路,以期获得种子中富含EPA和DHA的水稻新材料。人工合成优化后的EPA和DHA合成相关的7个基因,利用不同的水稻胚乳特异性高强度启动子,以及不依赖启动子的3’-非翻译区(untranslated regions, UTRs),分别与这些目的基因组合,利用高效多转基因堆叠系统II (transgene stacking II, TGS II)构建到植物表达载体中,通过农杆菌介导法进行遗传转化,分别获得8个(pEPA)和7个(pDHA)独立的转基因株系。PCR和Southern杂交分析,证明目标基因已经以不同的拷贝数整合到水稻基因组中。本研究成功构建了EPA和DHA合成途径植物表达载体,初步获得了阳性转基因植株,为后续ω-3 VLPUFAs在水稻种子中合成和积累的深入研究和应用打下了基础。

Eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)are the most nutritious omega-3 very long-chain polyunsaturated fatty acids(VLPUFAs),which mainly come from certain deep-sea fish oils,algae and microorganisms.Due to overfishing,heavy metal pollution and the high price of extraction,it is difficult to meet the market demand.There is no omega-3 VLPUFAs pathway in higher plants.Therefore,the development of new plant resources rich in EPA and DHA has broad application prospects.In this study,seven genes related to the synthesis of omega-3 VLPUFAs were introduced into rice to create a pathway for the synthesis of ω-3 VLPUFAs,in order to obtain new rice materials that are rich in EPA and DHA.Seven genes related to EPA and DHA synthesis were synthesized and combined with different rice endosperm specific high-strength promoters and 3'-untranslated regions(3'-UTRs)independent of promoters to construct plant expression vector via multigene assembly and transformation vector system(transgene stacking II,TGS II).Through A grobacterium-mediated transformation,regenerated plants were obtained and identified.Eight(pEPA)and seven(pDHA)transgenic lines were obtained.PCR and Southern blot analysis showed that the genes had been integrated into rice genome with different copy numbers in transgenic plants.In this study,the plant expression vectors of EPA and DHA synthesis pathway were successfully constructed,and the positive transgenic plants were initially obtained,which provide a foundation for further study and application on the synthesis and accumulation of omega-3 VLPUFAs in rice seeds.