胡麻脂肪酸去饱和酶基因(fads)差异表达分析

为了了解fad基因在胡麻蒴果发育过程中对不饱和脂肪酸的调控,对高、中、低三个不同亚麻酸(C18:3)含量的胡麻品种(’CDC Gold’,‘内亚7号’,’Linola’)进行了不同时期的品质测定,以及脂肪酸去饱和酶2a基因(fad2a)、脂肪酸去饱和酶2b基因(fad2b)、脂肪酸去饱和酶2c基因(fad2c)、脂肪酸去饱和酶3a基因(fad3a)、脂肪酸去饱和酶3b基因(fad3b)的qRT-PCR定量分析。结果表明,随着蒴果成熟,可溶性糖含量呈降低趋势,粗脂肪与粗蛋白不断积累,且差异显著(p<0.05)。fad2a基因、fad3a基因以及fad3b基因在各个时期中的表达符合正态分布。以0 d的蒴果为对照,在胡麻种子形成过程中,‘内亚7号’的三个基因在5 d和15 d的表达量迅速增加,到30 d时急剧减少,15 d的fad2a基因表达量为5.23倍,fad3a基因表达量是fad2a基因表达量的14.52倍,fad3b基因的表达量是fad2a基因表达量的16.14倍,表明这三个基因参与不饱和脂肪酸积累过程。在低亚麻酸含量品种‘Linola’30 d中,fad3a基因是fad2a基因表达量的52.71倍,fad3b呈下调趋势;在高亚麻酸含量品种‘CDCGold’30d中,fad3a基因与fad2a基因的表达量均呈下调趋势,fad3b的表达量为3.92倍;在中等亚麻酸含量品种‘内亚7号’中,fad2a基因表达量降低了0.31倍,fad3a基因是fad3b基因表达量的1.87倍。fad2b基因、fad2c基因熔解曲线不稳定,峰值低,可以在后续试验中继续探索。     

播娘蒿油酸脱氢酶基因(DsFAD6)的克隆和表达分析

采用RACE-PCR的技术克隆了播娘蒿的油酸脱氢酶基因(DsFAD6),它是催化油酸转化为亚油酸的一个关键酶基因.DsFAD6 cDNA全长序列的开放阅读框长1 344 bp,编码一个由447个氨基酸残基组成的蛋白质,推测的蛋白质相对分子质量为51.16 kD,等电点为9.27.序列分析表明DsFAD6的氨基酸序列具有3个高度保守的组氨酸盒子,该结构在去饱和酶中是高度保守的,且在N端预测到信号肽,初步认定该酶定位于质体.序列比对和系统发生分析显示DsFAD6与十字花科植物FAD6基因具有较高一致性.RT-PCR分析表明,DsFAD6在根、茎、叶、花蕾、花及幼嫩角果中均表达,但在茎、叶和幼嫩角果中表达较高.胁迫诱导表达中,DsFAD6在播娘蒿叶片中表达明显受伤害胁迫诱导,但在冷胁迫下呈负相关.     

植物中γ-亚麻酸合成关键酶——△^6-脂肪酸脱氢酶

γ-亚麻酸作为人体必需的不饱和脂肪酸，对人体的激素调节及脂肪酸代谢发挥着重要的生理作用。△^6-脂肪酸脱氢酶是多不饱和脂肪酸合成途径中的限速酶。本文介绍了不饱和脂肪酸γ-亚麻酸代谢途径中的关键酶△^6-脂肪酸脱氢酶的结构功能与目前△^6-脂肪酸脱氢酶的基因工程研究进展，并对其应用进行了展望，以期为利用基因工程手段生产γ-亚麻酸提供参考。     

Cloning, tissue distribution and hormonal regulation of stearoyl-CoA desaturase in tilapia, Oreochromis mossambicus

The stearoyl-CoA desaturase cDNA in tilapia (Oreochromis mossambicus) was cloned by RT-PCR and RACE, and it was compared with those in grass carp, common carp and milkfish. Nucleotide sequence analysis revealed that the full length of cDNA (1172 bp) clone encompasses 1008 bp open reading frame (ORF) encoding 336 amino acid residues. The deduced amino acid sequence shares 78–82% identity with the teleosts and 64–66% with mammals compared, and like these fish, the cloned tilapia stearoyl-CoA desaturase amino acid sequence conserves three histidine cluster motifs (one HXXXXH and two HXXHH), which functioned as non-heme iron binding sites, essential for stearoyl-CoA desaturase activity. RT-PCR and Northern blot analysis reveal that tilapia stearoyl-CoA desaturase is expressed only in liver, but the stearoyl-CoA desaturase expression in multiple tissues was observed in milkfish, grass carp and carp. Further, the hormonal regulation of stearoyl-CoA desaturase gene expression was investigated by a single injection of 17β-estradiol and testosterone. The results showed that the administration of 17β-estradiol to tilapia led to a greater increase in desaturase activity than testosterone, and higher doses of steroids produced greater increases in enzyme activity. The comparative RT-PCR analysis showed that the stearoyl-CoA desaturase mRNA level increased significantly in 17β-estradiol treated animals, especially in the groups receiving a single injection of 50 mg 17β-estradiol. This was reflected in the decrease in the saturated fatty acids and the increase in the monounsaturated fatty acids. The proportion of the polyunsaturated fatty acids was not affected.     

Effects of dietary n−3 polyunsaturated fatty acids on lipid and fatty acid composition and haematology of juvenile Arctic charr Salvelinus alpinus (L.)

The effect of dietary n−3 and n−6 polyunsaturated fatty acids (PUFAs) on juvenile Arctic charr Salvelinus alpinus (L.) were investigated with respect to essential fatty acid (EFA) deficiency and lipid metabolism using one commercial and 12 casein-based test diets. Arctic charr with mean weight of 1.6g were fed test diets for 12 weeks at 10°C. At the end of the feeding, blood, liver, muscle and whole fish were sampled to determine haematocrit, haemoglobin, water content, lipid and fatty acid composition. Charr fed diets containing 0–1.0% n−3 PUFAs showed typical EFA deficiency signs: fatty liver or elevated water content in whole body or substantial accumulation of 20:3n−9 in liver polar lipids. These signs were less apparent or disappeared when charr were fed diets containing ≥ 2.0% 18:3n−3. No correlation was found between dietary PUFAs and haematocrit or haemoglobin values. Significant changes in fatty acid composition of liver polar lipids in charr fed dietary PUFAs indicate that charr can convert 18:3n−3, 18:2n−6 and 20:5n−3 into long-chain PUFAs. While charr had a direct incorporation of dietary 22:6n−3 into liver and muscle there appears to be preferential utilization of n−3 PUFAs for elongation and desaturation. The conversion of 18:4n−3 was less in muscle than in livers. These findings, combined with data on growth and feed efficiency reported previously by Yang and Dick (1993), indicate that charr require 1−2% dietary 18:3n−3 (dry weight). Small amounts of dietary 18:2n−6 (up to 0.7%) did not have detrimental effects on charr.     

植物抗寒工程中脂肪酸去饱和酶研究进展

低温是影响植物生长的重要因素之一。本文介绍了多不饱和脂肪酸与植物抗寒性的正相关关系,并从脂酰-ACP去饱和酶、脂酰-CoA去饱和酶、脂酰-脂去饱和酶等几个方面综述了近几年里与植物抗寒性相关的脂肪酸去饱和酶基因的克隆、功能鉴定、表达调控等方面的研究进展。也讨论了一些对不饱和脂肪酸和植物耐低温间因果关系存在争议的研究报道。展望了应用基因工程技术培育出抗寒抗冻性较强的林业木材品种。     

棉花FAD 2-1基因的克隆及其ihpRNA和amiRNA干扰载体的构建

 采用RT-PCR方法克隆了棉花△-12脂肪酸去饱和酶基因(GhFAD2-1)的cDNA全长序列,该基因含有1158 bp的完整开放阅读框。棉花FAD2-1是种子中编码催化油酸形成不饱和脂肪酸的关键酶的基因,选取GhFAD2-1基因中的一段特异的515 bp片段,构建了种子特异性启动子NAPIN调控的ihpRNA干扰表达载体pFGC1008-NAPIN-FAD2-1,同时构建了针对GhFAD2-1基因的人工miRNA表达载体pCAMBIA1302- amiRNA-FAD2-1。     

棉花八氢番茄红素脱氢酶GhPDS1基因的克隆与表达谱分析

八氢番茄红素脱氢酶(PDS)在胡萝卜素生理代谢和病毒诱导外源基因沉默中起着非常重要的作用.本研究通过同源克隆方法,分析已报道植物八氢番茄红素脱氢酶氨基酸保守区,设计筒并引物,扩增出编码棉花PDS基因cDNA中间片段.通过RACE技术成功地克隆了棉花PDS基因的全长cDNA,命名为GhPDS1(GenBank No.HQ...