Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein.
Advanced engineering of lipid metabolism in Nicotiana benthamiana using a draft genome and the V2 viral silencing-suppressor protein.
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DOI：
10.1371/journal.pone.0052717
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Wood CC
中科院分区：
文献类型：
--
作者：
Naim F;Nakasugi K;Crowhurst RN;Hilario E;Zwart AB;Hellens RP;Taylor JM;Waterhouse PM;Wood CC
本塞姆氏烟草的瞬时叶片测定广泛应用于植物科学，其中一项应用是快速组装产生新脂肪酸谱的复杂多基因途径。如果能够同时过度表达转基因，同时准确沉默内基因，这种快速而简便的测定将得到进一步改进。在这里，我们报告了本塞姆氏烟草基因组资源草案，涵盖 3.1 Gb 单倍体基因组的 75% 以上。该资源揭示了 NbFAD2 家族的两个成员：NbFAD2.1 和 NbFAD2.2，定量 RT-PCR (qRT-PCR) 证实了它们在叶子中的表达。通过 qRT-PCR 和生化检测监测，使用发夹 RNAi 沉默 FAD2 活性。通过使用来自番茄黄曲叶病毒的替代病毒沉默抑制蛋白 V2，将内源 FAD2 活性的沉默与转基因的过度表达相结合。我们证明 V2 允许转基因最大限度地过度表达，但最重要的是，它还允许发夹 RNAi 不受阻碍地运行。为了说明基于 V2 的叶片测定系统的功效，将内源脂质从 18:1 的去饱和分流到以 18:1 作为底物开始的延伸反应。这些基于 V2 的叶片检测比基于 p19 的检测多产生了约 50% 的延长脂肪酸产物。对针对 NbFAD2 的发夹 RNAi 产生的小 RNA 群体的分析证实，siRNA 群体以源自发夹的 21 和 22 nt 物种为主。总的来说，这些新工具扩大了瞬时叶分析中代谢工程的用途和可能性。
The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18∶1 to elongation reactions beginning with 18∶1 as substrate. These V2-based leaf assays produced ∼50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays.
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DOI：
10.1023/b:anto.0000024910.57117.16
发表时间：
2004-08-01
影响因子：
2.6
作者：
Hilario, E;Buckley, TR;Young, JM
通讯作者：
Young, JM
影响因子：
11.6
作者：
Millar, AA;Wrischer, M;Kunst, L
通讯作者：
Kunst, L
影响因子：
7
作者：
Li, Ruiqiang;Zhu, Hongmei;Wang, Jun
通讯作者：
Wang, Jun
DOI：
10.1073/pnas.0706701104
发表时间：
2007-09-11
影响因子：
11.1
作者：
Brosnan, C. A.;Mitter, N.;Carroll, B. J.
通讯作者：
Carroll, B. J.
影响因子：
2.7
作者：
Carlsson, Anders S.;Yilmaz, Jenny Lindberg;Green, Allan G.;Stymne, Sten;Hofvander, Per
通讯作者：
Hofvander, Per