适于烟草脆裂病毒诱导的本氏烟基因沉默分析的对照载体构建(英文)

对烟草脆裂病毒(Tobacco rattle virus,TRV)诱导的基因沉默分析体系中目前最常用的对照载体——空pYL156载体(pYL156∷00)对沉默处理后番茄和本氏烟(Nicotiana benthamiana)植株的病毒症状和生长影响进行分析.结果表明:pYL156∷00沉默处理后的番茄和本氏烟植株均表现出严重的系统性病毒症状,生长受到显著抑制,因此,pYL156∷00并不是一个用于TRV诱导的番茄和本氏烟基因沉默分析的好的对照载体;为获得更好的对照载体,试验构建了2个新的对照载体pYL156∷NIRi和pYL156∷eGFP,它们分别通过在pYL156∷00载体多克隆位点插入253bp菜豆亚硝酸还原酶基因内含子序列和400bp eGFP基因片段而获得,对这2个对照载体对沉默处理后本氏烟植株的病毒症状和生长情况的影响与pYL156∷00进行比较分析.结果表明:pYL156∷00沉默处理后的植株表现出较严重的系统性病毒症状,生长受到显著抑制,其中26.7%植株死亡;pYL156∷NIRi沉默处理后的植株也表现出明显的病毒症状以及生长受抑制现象,但与pYL156∷00相比,所受影响较小,只有13.3%植株死亡;而pYL156∷eGFP沉默处理后的植株不表现明显的病毒症状,植株生长也不受显著影响;病毒积累量检测结果显示,3个对照载体沉默处理的植株病毒症状和生长受抑制情况与植株体内TRV病毒的积累水平密切相关.这些结果表明,新构建的pYL156∷eGFP可以作为一个优越的对照载体应用于TRV诱导的基因沉默分析.

Establishment of a suitable control vector for Tobacco rattle virus-induced gene silencing analysis in Nicotiana benthamiana

Effects of the empty pYL156 vector(pYL156∷00),the most commonly used control vector for Tobacco rattle virus(TRV)-induced gene silencing analysis on viral symptom development and growth of VIGS-treated tomato and Nicotiana benthamiana plants were investigated.It was shown that VIGS-treatment for pYL156∷00 caused severe systemic viral symptoms and obvious growth repression in treated plants.Therefore,pYL156∷00 is not a good control vector for TRV-induced gene silencing analysis in these plant species.To set a better control for this analysis,two new constructs,pYL156∷NIRi and pYL156∷eGFP,were made.They were released by inserting a 253 bp fragment of a Phaseolus vulgaris nitrite reductase gene intron and a 400 bp fragment of the jellyfish GFP coding sequence in pYL156∷00,respectively.Effects of the two new control constructs on viral symptom development and growth of VIGS-treated N.benthamiana plants were compared with pYL156∷00.The results showed that VIGS-treatment for pYL156∷00 caused severe systemic viral symptoms and significant growth repression in treated plants,resulting in death of 26.7% plants.VIGS-treatment for pYL156∷NIRi also led to obvious viral symptoms and growth repression,but in a less severe extent than that for pYL156∷00,resulting in death of 13.3% plants.However,VIGS-treatment for pYL156∷eGFP did not cause any obvious viral symptom and growth repression.Severity of viral symptom and growth repression was well correlated with the accumulation level of TRV virus.These results demonstrate that pYL156∷eGFP is an excellent control vector for TRV-induced gene silencing analysis,and provide some insights into the direction to establish an excellent control for VIGS analysis.