Virus-induced gene silencing in various Prunus species with the Apple latent spherical virus vector
Virus-induced gene silencing in various Prunus species with the Apple latent spherical virus vector
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DOI：
10.1016/j.scienta.2015.12.031
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发表时间：
2016-02
影响因子：
4.3
通讯作者：
T. Kawai;Ayako Gonoi;Michiya Nitta;N. Yamagishi;N. Yoshikawa;R. Tao
中科院分区：
文献类型：
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作者：
T. Kawai;Ayako Gonoi;Michiya Nitta;N. Yamagishi;N. Yoshikawa;R. Tao
病毒诱导的基因沉默（VIGS）是一种快速、有效的基因功能分析工具。我们先前报道了成功地诱导杏（Prunus armeniacaL.）内源八氢番茄红素脱饱和酶（PDS）基因的VIGS。利用苹果潜伏球形病毒（ALSV）载体。与此相反，我们试图感染日本杏（Prunus mumeSiebold & Zucc.）用ALSV载体介导的转座试验不成功，表明李属植物对ALSV的敏感性可能存在种和/或品种依赖性差异。在本研究中，我们研究了基于ALSV载体的VIGS基因评价系统是否适用于杏、甜樱桃（Prunus aviumL.）扁桃[Prunus dulcis（Mill.）D. A. Webb.]，桃（Prunus persicaBatsch），日本杏，日本李（Prunus salicinaLindl.），和欧洲李（Prunus domesticaL.）。利用本氏烟草（Nicotiana benthamiana）为材料，扩增了携带烟草PDS基因部分序列的ALSV载体，并利用基因枪法将其接种到李属植物幼苗的子叶中。典型的PDS沉默表型，其特征是上部叶片均匀变色，在甜樱桃和一些品种的杏和杏仁接种后几周观察。在感染的叶片中PDSmRNA的量显着减少，而21 nt的反义小RNA，这被认为是发挥核心作用的向导RNA在PDSmRNA降解，高度积累。但对日本杏、日本李、欧洲李等杏和扁桃品种的ALSV侵染均未成功。此外，虽然桃中ALSV的感染率很高，但在受感染的叶片中观察到严重的淡斑（病毒感染症状）。这些结果共同表明，ALSV感染和VIGS的效率可能会有所不同，这取决于物种和/或品种在李。并对ALSV介导的VIGS系统在李属植物基因功能研究中的应用进行了讨论。
Virus-induced gene silencing (VIGS) has been used as a rapid and effective tool for functional analysis of genes in various plants, including woody fruit tree species. We previously reported the successful induction of VIGS of the endogenousPHYTOENE DESATURASE(PDS) gene in apricot (Prunus armeniacaL.) usingApple latent spherical virus(ALSV) vectors. In contrast, our attempts to infect Japanese apricot (Prunus mumeSiebold & Zucc.) with ALSV vectors was unsuccessful, suggesting that species- and/or cultivar-dependent differences of ALSV susceptibility may exist inPrunus. In this study, we investigated whether this VIGS-based gene evaluation system using ALSV vectors was applicable to sevenPrunusspecies, including apricot, sweet cherry (Prunus aviumL.), almond [Prunus dulcis(Mill.) D. A. Webb.], peach (Prunus persicaBatsch), Japanese apricot, Japanese plum (Prunus salicinaLindl.), and European plum (Prunus domesticaL.). ALSV vectors carrying part of the apricotPDSgene sequence were amplified inNicotiana benthamiana, and inoculated into the cotyledons ofPrunusseedlings by particle bombardment. TypicalPDS-silenced phenotypes, characterized by uniform discoloration of the upper leaves, were observed in sweet cherry and some cultivars of apricot and almond several weeks after inoculation. The amounts ofPDSmRNA in the infected leaves were significantly reduced, while a 21 nt antisense small RNA, which was assumed to play a central role as a guide RNA inPDSmRNA degradation, was highly accumulated. However, ALSV infection of Japanese apricot, Japanese plum, European plum, and the other cultivars of apricot and almond was unsuccessful. Furthermore, although the infection rate of ALSV in peach was high, severe pale spots (a viral infection symptom) were observed in the infected leaves. These results collectively suggested that the efficiency of ALSV infection and VIGS could vary depending on species and/or cultivar inPrunus. The possible use of the ALSV-mediated VIGS system for functional analysis of genes inPrunusis discussed.