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油棕(Elaeis guineensis Jacq.)又名油椰子,为原产于热带非洲的棕榈科多年生单子叶木本植物[1],在中国主要分布于海南省南部、云南省西双版纳等地区,寿命长、经济价值高,年均单位面积产油量(CPO)高达 3.7 t·hm−2[2],是世界油料的重要来源之一。油棕果实的中果皮是重要的油脂积累组织,主要产物为棕榈油,棕榈油的用途广泛,在食品加工、轻工业、机械和生物清洁能源等领域均有应用[3-6]。棕榈油中饱和脂肪酸与不饱和脂肪酸的比例约为 1∶1 。新鲜油棕果肉含有约 38%~45% 的棕榈酸(C16:0),38%~44% 的油酸(C18:1),10%~12% 的亚油酸(C18:2)和 3%~5% 硬脂酸(C18:0),总饱和度约为 50%。提高食用油中油酸(C18:1)等不饱和脂肪酸含量,降低饱和脂肪酸在总油脂中的比例,一直是油棕遗传改良的重要目标之一。
乙酰基酰基载体蛋白硫酯酶(fatty acyl-ACP thioesterase, FAT)是一种由核基因编码的可溶性酶,主要作用于酰基和ACP间的硫酯键,水解硫酯键、释放ACP和游离的脂肪酸,从而停止碳链的延长[7-8]。目前研究的高等植物中酰基-ACP硫酯酶根据其分解底物的特异性可分为两类[9]:一类是FATA(酰基载体蛋白硫酯酶A),在油棕中主要作用于十八碳链脂肪酸链(C18:X-ACP),但在不同的物种中具体对应关系有较大差异;另一类则是FATB(酰基载体蛋白硫酯酶B),主要作用饱和脂酰基-ACP[10],对C16:0-ACP的催化活性最高[11]。
病毒诱导的基因沉默 (virus induced gene silencing, VIGS)利用插入了植物靶基因cDNA片段的病毒载体侵染植物,进入植株的病毒在复制和转录过程中能够特异性识别与插入片段序列同源的mRNA,使其降解从而抑制靶基因的表达[12]。常规遗传转化验证基因功能时,常需要植物生长至F2代甚至F3代时才能展开研究,而VIGS体系的应用,可在较短时间内通过观察植株的表型或生理指标的变化,应用VIGS技术对基因的功能进行验证。VIGS体系的应用特别适合于多年生木本植物中基因的功能鉴定[13]。在本研究中,油棕胚状体实验材料的选择可以克服油棕遗传转化周期长的困难,加快基因功能的鉴定与研究,同时保持了油棕的物种特性。
油棕是热带重要油料作物,产油量较高且用途广泛。然而,作为食用油来说,棕榈油脂肪酸比例的改善一直是油棕的重要育种目标之一[14-15]。其中,油棕乙酰基-ACP硫酯酶决定了脂肪酸链的长度和饱和度,被认为对脂肪酸组分的改善有重要的价值。目前,油棕中油棕乙酰基-ACP硫酯酶的具体功能尚不明确,沉默EgFATA基因的表达并测定基因沉默前后脂肪酸种类的变化,能够解析EgFATA的具体功能及其在脂肪酸调控中的作用。
Cloning and functional analysis of the fatty acyl-ACP thioesterase EgFATA in oil palm
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摘要: 为了探索油棕(Elaeis guineensis Jacq.)EgFATA基因的生物学功能,解析EgFATA基因在油棕中与脂肪酸积累的对应关系,为进一步研究EgFATA作用机制奠定基础,从油棕果cDNA中克隆得到了EgFATA基因的全长序列,经生物信息学分析发现,EgFATA编码区由1 155个碱基组成,可编码385 个氨基酸,相对分子量为91.89 kDa,与海藻PdFATA亲缘关系最近。在此基础上,构建EgFATA的病毒沉默载体并侵染油棕胚状体,RT-qPCR结果表明,EgFATA的表达量被下调85%~90%。同时,对基因沉默胚状体进行脂肪酸气相色谱法分析,硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)的含量均显著性降低。以上结果表明在油棕胚状体中,EgFATA对C18:0-ACP、C18:1-ACP、C18:2-ACP的水解具有催化作用,且对C18:1-ACP的催化活性最强。
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关键词:
- 油棕 /
- 乙酰基酰基载体蛋白硫酯酶 /
- 油脂代谢调控
Abstract: Oil palm (Elaeis guineensis Jacq.) is an important tropical palm with high oil production. Fatty acyl-ACP thioesterase (FAT) is the key enzyme for hydrolysis of acyl and ACP and termination of carbon chain extension. The oil palm FATA family mainly acts on eighteen-carbon chain fatty acids (18: X-ACP), but the specific hydrolysis substrates and preferences are unknown. In this study, the full-length sequence of the EgFATA gene was first cloned from oil palm fruit. Bioinformatics analysis indicated that the EgFATA coding region consists of 1155 bases and can encode 385 amino acids with a molecular weight of 91.89 kDa, and is most closely related to the alga PdFATA. A viral silencing vector for EgFATA was constructed and then infected the embryoids of oil palm, and RT-qPCR results showed that the expression of EgFATA was down regulated by 85%-90%. At the same time, fatty acid GC analysis of the silenced embryoids showed that the contents of stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) were significantly reduced. The aforesaid results indicated that EgFATA had catalytic effect on the hydrolysis of C18:0-ACP, C18:1-ACP and C18:2-ACP in the oil palm embryoids, with the highest catalytic activity observed in the hydrolysis of C18:1-ACP. This study initially explored the biological functions of EgFATA genes in oil palm, providing a theoretical basis for further exploration of the molecular mechanisms of EgFATA in regulation of lipid metabolism.-
Key words:
- oil palm /
- fatty acyl-ACP thioesterase /
- regulation of lipid metabolism
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