胡麻脂氧合酶基因LuLOX1的克隆与表达分析

Cloning and Expression Analysis of LuLOX1 Encoding a Lipoxygenase Gene in Flax (Linum usitatissimum L.)

胡麻的籽粒富含多不饱和脂肪酸-亚麻酸,易被脂氧合酶(LOX)氧化,导致油脂的酸败。因此,油脂的稳定性和风味是影响胡麻油质量的重要因素。脂氧合酶是一类非血红素双加氧酶,其催化不饱和脂肪酸如亚油酸和亚麻酸的氢过氧化反应。本研究旨在阐明在胡麻种子发育时期脂氧合酶基因(LuLOX1)的表达、酶活性与品质的关系。本试验利用基于亚麻全基因组DNA测序数据,对LuLOX1基因进行克隆,利用RT-PCR方法进行LuLOX1基因表达分析,采用分光光度计法进行LOX酶活性测定。结果显示,LuLOX1基因的cDNA全长为2 607 bp,编码868个氨基酸,LuLOX1蛋白分子质量和等电点分别为98.87 kD、6.36。LuLOX1在种子发育早期高表达,之后呈下降的趋势,成熟期几乎检测不到该基因的表达。LuLOX1的酶活性变化与LuLOX1基因表达模式一致。该研究结果表明,LuLOX1可能参与了种子发育过程中脂肪酸氧化的调控。本研究为进一步研究其功能,通过脂氧合酶调控改进胡麻油脂品质奠定了基础。

The seed oil of flax(Linum usitatissimum L.)is notable for its high level of polyunsaturated fatty acids including linolenic acid,which could be oxidized by lipoxygenase,which result in oxidative acidity of oils.Therefore,stability and off-flavors are vital characteristics that determine the quality of flax oil.Lipoxygenases(LOXs)are a class of non-heme iron containing dioxygenases which catalyze the hydroperoxidation of polyunsaturated fatty acids such as linoleic and linolenic acids.Experiments were conducted in order to elucidate the role of LOXs during seed development and in relation to seed oil quality.The full length cDNA of LuLOX1 was cloned based on flax whole genome DNA sequence database.The expression of LuLOX1 was determined by RT-PCR and the catalytic activity of LuLOX1 was determined by spectrophotometer.Results have shown that the CDNA of LuLOX1 gene was 2 607 bp,encoding a protein of 868 amino acids.The molecular weight and isoelectric point of LuLOX1 were 97.87 kD and 6.36,respectively.RT-PCR carried out with RNAs extracted from seeds at different development stages showed that LOX is mainly expressed at early developmental stages.The results were consistent with those of LOX activity assays.The patterns of LuLOX1 expression were consistent with LuLOX1 activities.This indicated that LuLOX1 might be involved in regulation of fatty acid oxidation during seed development.These results laid the foundation for further study on the function of LuLOX1 that may play an important role in enhancement of the quality of flaxseed oil through lipoxygenase.