花生AhFAD2基因抑制表达的转基因后代分析

Analysis of the peanut transgenic offspring with depressing AhFAD2 gene

摘要:

FAD2 (Δ12 fatty acid desaturase，Δ12FAD或FAD2) 是催化油酸在脂肪酸碳链 Δ12位脱氢生成亚油酸的关键酶。在花生中，FAD2酶活性下降或失活可提高籽粒中油酸的相对含量，改善花生籽粒及制品的品质和氧化稳定性。通过将种子特异性表达Lectin启动子和CaMV35S启动子驱动的倒位重复AhFAD2基因RNAi干扰结构转入花生，获得了以丰花1号 (FH1) 和花育23 (HY23) 为受体、携带上述2种转化结构、稳定遗传的花生转基因纯合体株系，转基因花生主要农艺性状与非转基因对照基本一致。实时荧光定量分析发现，各转基因株系发育种子中AhFAD2基因的转录水平普遍下调。气相色谱法进一步测定了部分转基因后代株系种子的脂肪酸含量及组成，籽粒中油酸含量分别平均提高了15.09% (HY23为受体)、36.40% (FH1为受体)，相应地，亚油酸含量平均下降了16.19%、29.81%，油亚比平均增加了38.02%、98.10%。各转基因株系的油酸含量显著提高；且在以FH1为受体的转基因株系后代籽粒以及种子特异性启动子驱动的转化结构中，RNAi的抑制效果更明显。通过RNAi技术抑制花生FAD2基因的表达，可以有效提高花生籽粒油酸含量。该技术体系可以为花生品质育种提供借鉴。

Abstract:

The delta-12 fatty acid desaturase (Δ12 FAD or FAD2) is a key enzyme that catalyzes oleic acid to linoleic acid by dehydrogenation at Δ12 position of fatty acid carbon chain. In peanut, reduction or loss of FAD2 activity could enhance the relative content of oleic acid in kernels, and improve the quality and oxidation stability of peanut kernels and products. RNA interference (RNAi) technology could lead to non-expression or down-regulated expression of AhFAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial AhFAD2 gene, which were driven separately by cauliflower mosaic virus (CaMV) 35S promoter or soybean agglutinin lectin seed-specific promoter. Homozygous transgenic lines carrying the two constructs stably in genetics were developed by peanut genetic transformation. There were no significant differences between the transgenic lines and the control through investigating the main agronomic traits. We analyzed the transcriptional level expression of AhFAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR (qRT-PCR). The results suggested that the target genes of transgenic lines were likely suppressed by RNA interference, but showed different transcriptional levels in different peanut transgenic lines. Compared with untransformed lines, the resulting down-regulation of AhFAD2 gene resulted in a 15.09% or 36.40% increase in oleic acid content in the seeds of transformed HY23 and FH1 lines respectively, and the content of linoleic acid decreased by 16.19% or 29.81%, correspondingly, the ratio of oleic acid and linoleic acid (O/L) improved by 38.02%, 98.10%. The oleic acid content had significant differences between the two transformation constructs, and also among different transgenic lines. Moreover, the inhibition effect of RNAi was more obvious in the transgenic lines with FH1 as the receptor, and with transformation structure driven by seed specific promoter. The suppressed expression of AhFAD2 gene enabled the development of peanut fatty acid, which indicated that RNA interference would be a reliable technique for the genetic modification of peanut seed quality and the potential for improvement of other traits as well.