特用油料作物紫苏α-亚麻酸合成调控机制研究

Perilla, a member of the mint family Lamiaceae, is a new oil crop with a unique medicine value for human health. Perilla seeds can accumulate 35-64% oil. The oil contains higher level of α-linolenic acid, a omega-3 fatty acid essentially required for human nutition and healthy. This special oil has been widely used in functional food, medicine, and oil industry. In the present project, multiple approaches including molecular biology and functional genomics tools will be employed for cloning and functional characterization of the key enzyme genes responsible for α-linolenic acid synthesis and accumulation in Perilla seeds. Diacylglycerol acyltransferase (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferase (PDAT) are the two enzymes that can catalyze the final acylation of triacylglycerol (TAG). This enzymic step has been proposed to be one of the rate-limiting steps in plant storage lipid accumulation and play an essential role in controlling both the quantitative and qualitative flux of fatty acids into storage TAGs. Our hypothesis is perilla DGAT and PDAT can specifically select α-linolenic acids and transfer the fatty acid into TAG. The Perilla mutant population (difference at seed oil and α-linolenic acid content) previously obtained by radiation treatment will be used as the genotype matterials to analyze DNA polymorphorism (such as SNP) of the target genes. An association analysis will be conducted to elucidate the relationship between the DNA sequence differences (mutant) and α-linolenic acid contents in seed oil. The functioal characterization the cDNA clones of the target genes will be examined in vivo by yeast expression system，the plant tissue instantaneous expression system and Arabidopsis genetic transformation system. The substrate specificity of the target enzymes cloned will be investigated in vitro using 14C labeled substrate (18:2-CoA, 1,2-dioleoyl-sn-glycerol, and so on). The present study will generate new knowledge for undestanding α-linolenic acid biosynthesis in perilla seeds, and provide novel gene elements and technology system for oil improvement in perilla and other oil crops by metabolic engineering.

紫苏[Perilla frutescens(L.)Var.crispa]是一种具有特殊医药价值的新型油料作物，紫苏籽油富含人体必须的α-亚麻酸（ALA），广泛用于功能性保健食品、药物及油脂化工业。本课题采用分子生物学和功能基因组学技术，以已创建的紫苏突变体群体和多种国内种质资源为试材，研究控制紫苏种子α-亚麻酸合成及积累的关键酶基因，即选择性转移α-亚麻酸于三酰基甘油脂(TAG)分子中的二酰甘油酰基转移酶(DGAT1、DGAT2)和磷脂二酰甘油酰基转移酶(PDAT）。分析这些酶基因的结构、多态性以及在种子发育过程中的表达模式与α-亚麻酸合成积累的相关性。应用同位素标记底物的体外生化测试，酵母表达体系，植物组织瞬时表达体系和拟南芥遗传转化体系鉴定这些酶基因的生物学功能和酶学特性。本研究为进一步培育种子富集α-亚麻酸的油料作物新品系提供新的基因元件和技术体系。

紫苏[Perilla frutescens (L.) Var. crispa]种子油富含α-亚麻酸，广泛用于功能性保健食品、药物及油脂化工业。本项目采用分子生物学和功能基因组学技术，研究了控制紫苏种子α-亚麻酸合成积累的关键酶基因，分析这些酶基因的结构、功能及在种子发育过程中的表达与α-亚麻酸合成积累的关系。气相色谱法对紫苏成熟种子及不同发育时期种子中脂肪酸组成成分、含量及其动态变化分析，结果表明：紫苏种子油的主要脂肪酸成分为硬脂酸(18:0)、棕榈酸(16:0)、油酸(18:1)、亚油酸(18:2) 和α-亚麻酸(18:3)。随着种子的发育亚油酸含量逐渐降低，而α-亚麻酸含量不断增加，至种子成熟时达总油脂的60%以上。采用电子克隆技术从晋苏1号发育种子中分离获得调控α-亚麻酸合成积累关键酶基因PfDGAT1/2和PfPDAT的cDNA克隆，并对其进行了分析。结果表明：PfDGAT2基因全长cDNA序列1249bp，其中包含990bp的开放性阅读框，共编码398个氨基酸。该基因在种子发育后期（即开花后30d）的表达量是开花后10d表达量的14.8倍；PfPDAT基因cDNA全长为2097bp，包含1554bp开放阅读框，共编码517个氨基酸。该基因在不同品种中主要于种子发育前期（即开花后10d或20d）高表达。应用酵母体系分别表达PfDGAT1/2和PfPDAT的cDNA克隆， 提取纯化微体蛋白，[14C]同位素标记和体外生化检测实验鉴定紫苏PfDGAT1/2和PfPDAT 酶学特征。结果显示：PfDGAT2和PfPDAT对底物α-亚麻酸有较强的选择性，主要控制α-亚麻酸积累，而PfDGAT1主要负责其他脂肪酸积累。转录组测序结果总碱基数达4.77 Gb，组装后获得160,584个Contigs和65,013条Unigene。其中45,679条Unigene获得了功能注释，占总数的70.3%。初步鉴定有2900个Unigene参与油脂代谢，并且有114个Unigene参与α-亚麻酸代谢。表达谱分析显示，开花后30d和40d种子间差异表达基因为3512个，其中1792个为上调表达基因，1720为下调表达基因。本研究为全面解析植物种子油脂及目标脂肪酸生物合成积累机制建立了新知识，亦为紫苏种子油脂品质遗传改良提供了理论和技术。