摘要
FAD2(Δ^(12) fatty acid desaturase,Δ^(12)FAD或FAD2)是催化油酸在脂肪酸碳链Δ^(12)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Ca MV35S启动子驱动的倒位重复Ah FAD2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Ah FAD2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。
The delta-12 fatty acid desaturase（Δ^12 FAD or FAD2） is a key enzyme that catalyzes oleic acid to linoleic acid bydehydrogenation at Δ^12 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 Ah FAD2 gene. We constructed two RNA interference expression vectors with the inverted repeat sequence of partial Ah FAD2 gene, which were driven separately by cauliflower mosaic virus（Ca MV） 35 S 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 Ah FAD2 gene in transgenic lines and the control by real-time fluorescence quantitative PCR（q RT-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 Ah FAD2 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 Ah FAD2 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.
作者
徐平丽
唐桂英
毕玉平
柳展基
单雷
Pingli Xu;Guiying Tang;Yuping Bi;Zhanji Liu;and Lei Shan(Bio-Tech Research Center,Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China;Shandong Provincial Key Laboratory of Crop Genetic Improvement,Ecology and Physiology,Jinan 250100,Shandong,China;Shandong Cotton Research Center,Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2018年第9期1469-1477,共9页
Chinese Journal of Biotechnology
基金
国家自然科学基金(Nos.31201272
31470349)
山东省农业良种工程(2014-2017)资助~~
关键词
花生
Δ^12脂肪酸脱氢酶
RNA干扰
转基因
油酸与亚油酸比值
peanut （Arachis hypogaea L.）
Δ^12fatty acid desaturase （FAD2）
RNA interference （RNAi）
transgenic
the ratio of oleic acid and linoleic acid （O/L）
