RNAi沉默转基因油菜fad2基因表达的种子特异性分析

Analysis of Seed-specificity of RNAi Silencing the fad2 Gene Expression of Transgenic Rapeseed(Brassica napus)

为研究RNAi沉默转基因油菜fad2基因表达的效果及其器官特异性,实时荧光定量PCR法分析比较了转基因油菜W-4与非转基因对照Westar开花后第7天、第14天、第21天、第28天的种子以及越冬期油菜根、茎、叶器官中的fad2基因的相对表达量。结果显示:对照Westsr种子中fad2基因的相对表达量随开花后的天数呈逐渐增加的趋势,但W-4种子中fad2基因的相对表达量在开花后第21天、第28天较对照Westar显著下降,降幅达到60%。说明转基因油菜W-4在种子发育过程中表达的fad2基因的dsRNA干扰了fad2基因表达。W-4与Westar在越冬期根、茎、叶器官中fad2基因的相对表达量基本一致,无显著性差异。脂肪酸组成分析结果显示:W-4种子中油酸平均含量达到81.5%较Westar增加了近15%,而多不饱和脂肪酸平均含量为5.25%,较Westar下降了近70%;但根、茎、叶中的脂肪酸含量二者无显著差异;结果表明转基因油菜中RNAi干扰fad2基因表达具有种子特异性,不干扰其它器官的fad2基因表达。研究还发现转基因油菜fad2基因表达下降的时期与napin基因表达时期同步,均始于开花后21d左右,表明目标基因的表达受napin启动子的准确控制。

In order to study the efficiency and specificity of RNAi silencing the expression of the endogenous fad2 gene of the transgenic line W-4, the relative expression of fad2 gene in seeds at different developmental stages of 7^th, 14^th, 21^th and 28^th day after flowering （DAF） as well as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and the non-transgenic control Westar by the quantitative real-time PCR. The results showed the relative expression of fad2 gene was gradually increasing with the days after flowering in the seeds of the control Westar, but the relative expression of fad2 gene in the seeds of the line W-4 was found decreasing significantly since the 21~ DAF and the decline was up to 60% compared with the control Westar. However, no significant difference in the relative expression of the fad2 gene in the root, stem and leave respectively was measured between the transgenic rapeseed and the non-transgenic control. Fatty acid composition analysis showed the oleic acid content in seeds of the line W-4 was 81.5% in average, 15% higher than that of the control, while polyunsaturated fatty acids was only 5.25% in average decreased nearly 70% compared with the control, but no significant difference in the seedling root, stem and leave of both the transgenic rapeseed and the control. The above results validated that RNA interference in transgenic rapeseed W-4 is of seed-specific, does not interfere with the fad2 gene expression in organs such as the root, stem and leave. The study also found that the period of the decline of the fad2 gene expression was well coincided with the expression of napin gene, both occurred at the 21^th DAF, indicating that the expression ofdsRNA of fod2 gene was accurately controlled by the napin promoter.