牡丹PsbZIP44转录因子调控α-亚麻酸合成的分子机制

Tree peony (Paeonia suffruticosa Andrews), a unique woody oil crop in China, contains dark oval seeds characterized by abundant unsaturated fatty acids (92.1%) and a high proportion of α-linolenic acid (ALA, 45.4%). The oil was proved to be a top-grade source for edible oil and nutritional supplements with great potential. ALA, an essential fatty acid, exerts a wide range of biological activities (e.g., as a nootropic) and prophylactic effects (including anti-inflammatory, antihyperlipidemic, anti-thrombotic, and anti-hypertensive). Previous researches revealed that PsFAD3 is the key structural gene in the biosynthesis of ALA, and the promoter of PsFAD3 contains two G-box cis-acting elements. We have identified a bZIP transcription factor PsbZIP44, and its spatio-temporal expression pattern is consistent with that of PsFAD3. In this study, we will analyze the binding activity of PsbZIP44 and its interacted protein factors with PsFAD3 promoter and transcriptional activation to understand the directly regulatory role and mechanism of PsbZIP44 and its complex on PsFAD3 gene expression. Additionally, the function of PsbZIP44 and its complex will be identified using overexpression and RNA interference technology in tree peony and transgenic technology in P. lactiflora. These data will lay the foundation for the breeding of tree peony cultivars with high ALA content and improving germplasm of other oil crops.

油用牡丹是我国特有的木本油料作物，种子中不饱和脂肪酸含量高达92.1%，尤其富含α-亚麻酸（ALA，45.4%），极具产业开发前景。ALA是人体必需脂肪酸，具有增强智力、抗炎症、降血脂、抗血栓和降血压等功能，对于人体健康具有重要意义。申请者前期研究阐明了PsFAD3是调控ALA合成的关键结构基因，同时发现其启动子区域含有2个G-box顺式元件。近期筛选到了一个与PsFAD3时空表达模式一致的转录因子PsbZIP44。本研究拟分析PsbZIP44及其互作的蛋白因子与PsFAD3启动子的结合以及转录激活活性，以明确PsbZIP44及其复合体对PsFAD3基因表达的直接调控作用及分子机制；拟通过牡丹体内瞬时过表达和基因沉默技术、芍药转基因技术明确PsbZIP44及其复合体在ALA合成过程中的作用。本项目的实施，将为油用牡丹分子育种提供重要依据，也可为其他油料作物的种质创新奠定基础。

油用牡丹为多年生木本植物，具有重要的观赏、药用和油用价值。其种子富含α-亚麻酸，是极具前景的木本油料作物之一。申请者在前期种质资源评价和转录组分析的基础上，克隆了两个牡丹品种‘凤丹白’和‘赛贵妃’中6个与调控PoFAD3基因表达相关的转录因子，亚细胞定位分析发现PobZIP44、PobZIP9和PoOBP3定位于细胞核。利用拟南芥原生质体验证了6个转录因子的转录激活活性，其中，PobZIP44具有显著的自激活活性；酵母单杂交和EMSA结果表明，PobZIP44能够结合PsFAD3基因启动子部分的G-box位点，从而激活其表达。通过PoFAD3启动子的截短和点突变验证，推断PobZIP44对PoFAD3启动子的激活依赖于与启动子上三个顺式元件的互作。为了进一步证实PsbZIP44基因在植物体内的功能，进行了拟南芥异源过表达及VIGS介导牡丹种子瞬时沉默，实验结果均证明PsbZIP44对于牡丹种子α-亚麻酸合成起到促进作用。此外，PobZIP44与其他转录因子的原生质体共转实验结果显示，PobZIP9会抑制PobZIP44的转录活性，自激活实验证实该抑制无需直接结合启动子，可能是通过与PobZIP44互作实现的。BiFC实验与PobZIP9突变蛋白实验的结果证实了PobZIP44与PobZIP9之间的互作，该互作依赖于PobZIP9的亮氨酸拉链区。本研究明确了PsbZIP44及其复合体在α-亚麻酸合成中的作用，为高α-亚麻酸含量的牡丹新品种的改良和选育提供了基础。