RNAi干扰油菜Δ^(12)-脂肪酸脱饱和酶基因的表达效果

Introduction of FAD2 gene fragment into Brassica napus via RNAi plasmid and enhanced oleic acid composition

为进一步研究RNA i介导的Δ12-脂肪酸脱饱和酶(FAD2)干扰基因对油菜内源FAD2基因表达的影响,以油菜肌动蛋白(-βActin)基因为内参照基因,提取转基因油菜幼嫩种子总RNA,通过半定量RT-PCR检测内源FAD2基因的相对表达量。结果表明,T1,T3代转基因种子中FAD2基因的相对表达量与对照相比明显降低。对T3代种子的油酸含量的分析表明,转基因油菜种子中油酸的含量比野生型增加了13.90到32.20个百分点,直接说明RNA i干扰体下调了FAD2基因的表达。因此,种子内源表达的FAD2基因被RNA i干扰体有效沉默,并且产生了能够稳定遗传两代的表型变化。

To further study the expression of △^12-fatty acid desaturase （FAD2） with RNAi mediated gene silencing, the total RNA of transgenic Brassica napus L. young seeds was extracted. Semi-quantitative RT-PCR was used to analyze the efficiency of RNAi construct in silencing the expression of the endogenous FAD2 gene, with β-Actin gene as the inner control. The results showed that the expression level of FAD2 gene in B. napus T1 and T3 transgenic seeds obviously decreased, but no change was found between the positive and negative controls. Using Agilent 6890N GC analyzer, the oleic acid content of T3 seed was examined. Oleic acid content of centigrade increased from 13.90 to 32.20 in the seeds of the transgenic plants compared with wild type, which indicated that RNAi interfered and reduced the expression of FAD2 gene. It demonstrated that the endogenous seed-expressed FAD2 gene was efficiently silenced by RNAi construct, and the phenotypic changes were inherited stably throughout two generations.