High rates of virus- induced gene silencing by tobacco rattle virus in Populus
High rates of virus- induced gene silencing by tobacco rattle virus in Populus
复制标题
杨树中烟草脆裂病毒引起的病毒诱导基因沉默率较高
DOI：
10.1093/treephys/tpv064
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发表时间：
2015-09-01
期刊：
影响因子：
4
通讯作者：
Chen, Shaoliang
中科院分区：
文献类型：
--
作者：
Shen, Zedan;Sun, Jian;Chen, Shaoliang
病毒诱导的基因沉默(VIGS)已被证明是研究草本植物基因功能的有效工具，但很少在树木上进行测试。对于木本植物来说，建立快速、可靠的转化体系尤为重要，因为许多木本植物对转化具有顽固性。在本研究中，我们建立了基于烟草Rattle病毒(TRV)的胡杨和加拿大杨两种杨树的VIGS系统。在这里，携带植烯脱饱和酶(PDS)基因的266aEuro...bp或558aEuro...bp片段的TRV构建体被农杆菌渗透到这两个杨树种的叶片中。农杆菌介导法将较短的插入片段TRV2-PePDS(266)导入寄主杨树，在两个树种中都产生了预期的光漂白，但较长的插入片段PePDS(558)则没有。VIGS的效率与温度有关，温度从18℃升高到28℃时，VIGS的效率增加。在28℃的温度下，优化的TRV-VIGS系统在两个受试杨树上产生了高达65-73和83-94%的沉默频率和效率。此外，与直接注射、喷雾和滴灌等其他农业入渗方法相比，注射器接种农杆菌在100aEul.mM乙酰丁香酮溶液中对这两种杨树的沉默效果更好。植物对VIGS的反应存在与物种相关的差异，这是因为P.aEuro…xaEuro...Canescens的光漂白症状比P.此外，经VIGS处理的胡杨在感染病毒数周后的恢复率(50%)高于感染TRV的胡杨(20%)。对参比基因的表达稳定性进行了筛选，以评估VIGS处理的胡杨和黄瓜中PePDS mRNA的相对丰度。PACT7在胡杨中稳定表达，通过三种不同的统计方法(geNorm、NormFinder和BestKeeper)选择UBQ-L作为P.aEuro…xaEuro...canescens最适合的参考基因。实时荧光定量聚合酶链式反应显示，两种杨树光漂白处理的叶片中PDS转录本显著减少(55-%)。我们的结果表明，基于TRV的VIGS为杨树基因功能分析提供了一个实用的工具，特别是对于那些在其他方面不能转化的杨树物种。
Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P.aEuro...xaEuro...canescens. Here, TRV constructs carrying a 266aEuro...bp or a 558aEuro...bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium-infiltrated into leaves of the two poplar species. Agrobacterium-mediated delivery of the shorter insert, TRV2-PePDS(266), into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS(558). The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28aEuro...A degrees C. The optimized TRV-VIGS system at 28aEuro...A degrees C resulted in a high silencing frequency and efficiency up to 65-73 and 83-94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100aEuro...mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P.aEuro...xaEuro...canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P.aEuro...xaEuro...canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P.aEuro...xaEuro...canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P.aEuro...xaEuro...canescens using three different statistical approaches, geNorm, NormFinder and BestKeeper. Quantitative real-time PCR showed significant reductions in PDS transcripts (55-64%) in the photobleached leaves of both VIGS-treated poplar species. Our results demonstrate that the TRV-based VIGS provides a practical tool for gene functional analysis in Populus sp., especially in those poplar species which are otherwise recalcitrant to transformation.