Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.
Efficient gene silencing mediated by tobacco rattle virus in an emerging model plant physalis.
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DOI：
10.1371/journal.pone.0085534
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
He C
中科院分区：
文献类型：
--
作者：
Zhang JS;Zhao J;Zhang S;He C
酸浆的果实有一个浆果和一个新奇的称为膨胀花萼综合征（ICS，也被称为“中国灯笼”）。阐明果实多样性的潜在发育机制需要一个有效的基因功能推理平台。本研究利用烟草脆裂病毒（TRV）介导的基因沉默系统对佛罗里达酸浆进行了研究。首先，我们确定了一个八氢番茄红素去饱和酶在佛罗里达州（PfPDS）的推定基因。用PfPDS-TRV载体感染酸浆属幼苗的叶导致漂白的植物，包括发育中的叶、花器官、ICS、浆果和种子。这些结果表明，局部VIGS处理可以有效地诱导系统性突变表型。qRT-PCR分析显示，漂白程度与内源性PfPDS的mRNA减少相关。多个渗透和生长协议的详细比较，使我们能够确定VIGS操纵在酸浆的最佳方法。随后，我们利用这种优化的VIGS方法下调两个MADS盒基因MPF 2和MPF 3的表达，并将所产生的效果与RNA干扰（RNAi）方法介导的基因下调进行比较。发现VIGS介导的基因敲低植物类似于MPF 2和MPF 3的RNAi转基因植物的花萼、果萼和花粉成熟的突变表型。此外，这两个MADS-box基因似乎在佛州海棠花梗发育中具有新的作用。与RNAi相比，基于VIGS的基因敲除的主要优点在于节省时间和易于操作的实用方面。尽管在一些器官中观察到的嵌合突变表型和遗传性缺乏，但TRV介导的基因沉默系统提供了一种替代有效的方式来推断酸浆属植物不同发育过程中的基因功能，从而有助于理解茄科植物形态多样性进化和发展的遗传基础。
The fruit of Physalis has a berry and a novelty called inflated calyx syndrome (ICS, also named the ‘Chinese lantern’). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in Physalis floridana. First, we characterized the putative gene of a phytoene desaturase in P. floridana (PfPDS). Infecting the leaves of the Physalis seedlings with the PfPDS-TRV vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous PfPDS. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in Physalis. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, MPF2 and MPF3, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both MPF2 and MPF3. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in P. floridana. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in Physalis, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.
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影响因子：
7.2
作者：
Ratcliff, F;Martin-Hernandez, AM;Baulcombe, DC
通讯作者：
Baulcombe, DC
影响因子：
7.2
作者：
Brigneti, G;Martín-Hernández, AM;Jones, JDG
通讯作者：
Jones, JDG
影响因子：
7.4
作者：
Burch-Smith, Tessa M.;Schiff, Michael;Dinesh-Kumar, S. P.
通讯作者：
Dinesh-Kumar, S. P.
影响因子：
56.9
作者：
Alonso, JM;Stepanova, AN;Ecker, JR
通讯作者：
Ecker, JR
影响因子：
7.4
作者：
Orzaez, D;Mirabel, S;Granell, A
通讯作者：
Granell, A