牡丹籽油α亚麻酸合成关键PsFADs基因的挖掘及作用机制研究

Peony seed oil contains a lot of unsaturated fatty acids and favored by consumers. The abundant α linolenic acid (C18:3) brings a great nutrition to human health. FADs, the fatty acid desaturase, control the key steps of oleic acid transfer to the linoleic acid and α linolenic acid. It is still unclear the regulation mechanism of synthesis of α linolenic acid. We found that the main unsaturated fatty acids were accumulated with the development of seeds. Eight FADs genes were cloned and found expressed differently at different stage. Therefore, we intended to confirm the key FAD genes which could regulate the synthesis of α linolenic acid to further complete the mechanism. This research will focus on fad mutation complementary experiment, FADs over-expression in tobacco seeds, and FADs transformation in Rhodotorula glutinis, FADs in vitro purification in E.coli and enzyme kinetics studies. At the same time, yeast cDNA library will be built and used for yeast one-hybrid screening to find the key transcription factors. Through the above two aspects, we will reveal the regulation mechanism of different FAD genes in α linolenic acid synthesis pathway and can dig out the key FAD genes and important FAD transcription factors.

已发现牡丹籽油因其含有大量不饱和脂肪酸而受到消费者的青睐，其中α亚麻酸（C18:3）含量高且对人体健康意义重大。FADs是控制油酸向亚油酸、α亚麻酸转化的关键脂肪酸脱氢酶，目前尚不清楚牡丹籽油中α亚麻酸合成的调控机制。申请者前期研究发现牡丹籽油含油量及主要不饱和脂肪酸含量随着种子成熟而不断积累，已获得的8个FADs基因在种子不同发育时期的表达存在差异，因此拟挖掘影响α亚麻酸合成的关键FADs基因，进一步阐释作用机制。本研究将进行FADs基因的拟南芥突变体互补、烟草超表达及黏红酵母的遗传转化分析其基因功能，结合原核体外表达FADs蛋白进行酶动力学研究；同时构建酵母cDNA文库，通过酵母单杂交筛选调控关键FADs基因的重要转录因子。通过以上两条途径相结合，揭示不同FAD基因在牡丹籽油α亚麻酸合成中的作用机制，挖掘出关键FAD基因及调控其表达的重要转录因子。

本研究以牡丹种子为研究材料，将PsFADs基因的时空表达与PUFA的积累规律进行关联，结合亚细胞定位结果，筛选出种子中特异表达的PsFAD2-1和PsFAD3-2两个重要成员。采用无细胞体系对膜蛋白PsFAD3-2进行体外表达，western blot结果显示成功。对烟草进行异源遗传转化，对超表达PsFAD3-2植株叶片中的PUFA进行GC-MS分析，发现C18:3含量显著上升，说明PsFAD3-2在α亚麻酸的合成催化中行使重要功能。通过Genome walking获得了PsFAD3-2上游1kb的启动子序列，并成功构建了牡丹cDNA酵母单杂交文库，酵母单杂交结果显示，IAA3生长素转录调控因子和SIWRKY40转录因子能够对PsFAD3-2启动子具有特异结合，在其转录调控中发挥潜在功能。本研究确定了影响牡丹籽油α亚麻酸合成的关键PsFAD3-2基因，并且进一步明确了其作用机制。本项目的完成不仅明确了不同FADs在α亚麻酸合成中是否行使一定功能、具有何种功能，同时还可为今后利用基因工程技术选育新品种、丰富现有油用牡丹品种奠定理论基础。