棉花种子ω-7脂肪酸生物合成途径的代谢组装

Omega-7 fatty acids including palmitoleic (16:1Δ9) and vaccenic (18:1Δ11) acids are important contributors to human health and are highly valued in pharmaceutical and industry applications. Due to the higher antioxidant and anti-cold properties, those unusual fatty acids are the desirable lipid materials for high-quality biodiesel production. Normal cotton seed oils contain none of this kind of fatty acids but higher level of palmitic acid(16;0). Palmitic acid is the precursor of ω-7 fatty acids. The chemical-physical features and physiological functions of palmitic acid are contrary to ω-7 fatty acids, limiting cotton oil's applications for human food and industry. Previously, we isolated several cDNA clones of the key enzymes and related genes. In this study, an integrative aprroach is developed to characterize biological functions of the genes cloned, particularly the substrate specificity of the enzymes including CoA-Δ9 desaturases and ACP-Δ9 desaturase. Metabolic engineering strategy is employed to assembly the biosynthesis pathway of ω-7 fatty acids in cotton seeds, and to investigate the regulatory mechanism responsible for lipid and oil synthesis in seeds. By doing these, this study will develop a new-type of cotton seed oil which can highly accumulate ω-7 fatty acids and simultaneously reduce palmitic acid level so as to provide a novel cotton oil variety for human food and industry including high-quality biodiesel production. In addition, the study will generates a number of new knowleges and techniques in the field of the regulation and gene modifications of plant lipid and oil metabolism, which can be further applied in genetic improvements of cottom amd other established oil crops.

ω-7脂肪酸包括棕榈油酸（16:1Δ9）和顺式-11-十八碳烯酸（18:1Δ11）等，不仅是一种健康有益型脂肪酸，而且由于具有较高的抗氧化和抗低温特性，是生产生物柴油的最佳脂肪酸。棉子油几乎不含ω-7脂肪酸, 却含有较高的棕榈酸(16:0)。棕榈酸是合成ω-7脂肪酸的前体，其理化性和生理功能与ω-7脂肪酸相反，因而限制了棉子油的营养、医药和工业应用。我们先期已获得催化棕榈酸生成ω-7脂肪酸的关键酶（CoA-Δ9D和ACP-Δ9D）及相关基因的cDNA克隆。本项目综合应用多种策略鉴定这些酶基因生物学功能及底物特异性，采用代谢工程技术在棉子中组装完整的ω-7脂肪酸生物合成途径，解析油脂代谢调控机制，以期培育富含ω-7脂肪酸、且棕榈酸显著降低的棉子油新种质，进而提高棉花油脂产品的经济效益。本项目可获得棉子油脂代谢调控和基因修饰新知识及新技术，进一步可应用于棉花及其他油料作物的遗传改良。

棉籽高含亚油酸（18:2; >50%）和棕榈酸（16:0; ～25%）导致棉籽油易氧化和对人体健康不利，所制取的生物柴油抗氧化性低和不耐低温。棕榈油酸（16:1Δ9）等ω-7脂肪酸是一类珍稀脂肪酸，具有极高的营养、医药以及工业价值。这类脂肪酸大多在一些野生植物中高水平积累，而在棉花等普通油料作物中极少合成。本课题旨在棉花发育中组装ω-7脂肪酸合成途径，以期获得棉籽油富集ω-7脂肪酸的新种质。 酰基-Δ9脱氢酶（Δ9D）是催化棕榈酸形成棕榈油酸的关键酶。棉花内源acyl-Δ9D家族全基因组鉴定显示，17个GhΔ9D成员中仅有2个（GhA-SAD8和GhD-SAD6）在各组织中高水平表达，且对18:0有底物选择性。因此，棉花种子本身缺失ω-7脂肪酸合成途径。离体及活体分析证实，来自野生植物富集ω-7脂肪酸种子的MucACP-∆9D和MiCoA-∆9D酶活性高、且对16:0有底物特异性。在棉花发育种子中特异表达这两个acyl-∆9D, 分别在质体和内质网上催化16:0生成16:1Δ9。共表达的转化体棉籽中新合成积累高达25%的ω-7脂肪酸，表明ω-7脂肪酸合成途径在棉子中组装成功。这些富含ω-7脂肪酸株系种子棕榈酸和亚油酸含量显著降低，其它农艺性状正常。应用Crispr-Case9分别敲除棉花内源GhFatB（酰基载体蛋白硫脂酶）和GhKASII（3-酮酯酰载体蛋白合成酶2）基因, 阻断16:0从质体转出和在质体内阻断ACP-16:0 延长为ACP-18:0, 从而使质体内ACP-Δ9脱氢酶作用的底物 ACP-16:0增加。分别将这两个基因敲除系与种子特异超表达MucACP-∆9D株系结合形成两个基因修饰的转基因株系。与仅表达MucACP-∆9D株系相比，GhFatB或GhKASII敲除和MucACP-∆9D超表达结合的株系16:1Δ9的合成量明显增加。显然，结合超表达16:0特异acyl-∆9D和敲除GhFatB及GhKASII可进一步提高棉籽中ω-7脂肪酸合成积累量。研究获得的这些转基因材料，可用于深入解析棉子油脂合成机制及代谢调控网络。 富含ω-7脂肪酸且农艺性状良好的工程株系可用于商业化生产高附加值的棉籽油，亦可进一步优化组装，培育ω-7脂肪酸含量更高的专用优异新种质。研究建立的技术策略也可用于其他油料作物的油脂代谢工程和优异性状聚合。