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小麦(Triticum aestivum)种子中脂氧合酶(lipoxygenase,LOX)可催化不饱和脂肪酸的氧化作用,在种子储藏期间产生氢过氧化物,导致小麦种子在储藏过程中种子活力和营养品质降低。因此,选育种子LOX活性低的小麦品种是小麦遗传改良的任务之一。为了获得种子脂氧合酶活性显著降低、外源Lox RNAi构件(Lox gene RNAi construct,Loxi)能稳定遗传且种子耐储藏性增强的转基因小麦新种质,本研究以小麦西农889、闫麦8911和陕优225转Loxi稳定遗传的TF7代转基因株系为材料,对转基因种子中Lox基因家族进行了半定量RT-PCR分析,结果表明,外源Loxi可有效抑制小麦籽粒中小麦脂氧合酶1(Triticum aestivum lipoxygenase 1,Ta Lox1)基因的表达,但小麦脂氧合酶2(Triticum aestivum lipoxygenase 2,Ta Lox2)和小麦脂氧合酶3(Triticum aestivum lipoxygenase 3,Ta Lox3)基因的表达量不变;对3个亲本及16个TF7代转基因株系的种子脂氧合酶活性检测,结果显示,与其亲本相比,转基因株系种子脂氧合酶活性显著降低;对人工加速老化处理7和14 d的转基因和对照种子分析发现,随着老化天数的增加种子活力降低,但转基因株系种子活力均显著高于其亲本材料;人工加速老化后脂肪酸组分和含量测定分析表明,转基因株系脂肪酸比其亲本材料氧化损失少。以上结果说明,转基因株系中Lox基因的RNAi可有效抑制其种子LOX活性,使转基因小麦后代种子耐储藏性增强;筛选鉴定得到的种子LOX活性显著降低且耐储藏性增强的转基因小麦株系可为小麦育种提供新的材料。
Lipoxygenase (LOX) in seeds of wheat (Triticum aestivum) can catalyze the oxidation of unsaturated fatty acids and produce hydroperoxides during the storage of seeds, resulting in reduced seed vigor and nutritional quality of wheat seeds during storage. Therefore, breeding wheat with low LOX activity is one of the tasks of wheat genetic improvement. In order to obtain a significant reduction of seed lipoxygenase activity, a new genetically modified wheat (Triticum aestivum L.) germplasm with stable genetic inheritance and enhanced seed storage resistance of Lox gene RNAi construct (Loxi) 8911 and Shan You 225 transgenic Loxi stable transgenic TF7 generation of transgenic lines as materials, semi-quantitative RT-PCR analysis of Lox gene family in transgenic seeds showed that exogenous Loxi can effectively inhibit wheat lipid lipid (Triticum aestivum lipoxygenase 1, Ta Lox1), but the expression of Triticum aestivum lipoxygenase 2 (Ta Lox2) and Triticum aestivum lipoxygenase 3 (Ta Lox3) The results showed that compared with their parents, the activity of lipoxygenase in the seed of the transgenic lines was significantly decreased. The effect of artificial accelerated aging treatment on the seed lipoxygenase activity of three transgenic lines and 16 TF7 transgenic lines was also studied. Analysis of transgenic and control seeds at 7 and 14 d showed that the seed vigor of the transgenic lines was significantly higher than that of the parental material as the aging days decreased, Determination of points and analysis showed that the transgenic lines which is less than the parent fatty acid oxidation loss of this material. The above results indicated that the RNAi of Lox gene in transgenic lines can effectively inhibit the LOX activity of the seed and enhance the seed storage resistance of transgenic wheat progenies. The transgenic wheat lines with significantly lower LOX activity and enhanced storage resistance were screened and identified Provide new material for wheat breeding.