CAJ | 학술논문

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栽体；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253 bp菜豆亚硝酸还原酶基因内含子序列和400 bp 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.
대연초취렬병독(Tobacco rattle virus,TRV)유도적기인침묵분석체계중목전최상용적대조재체——공pYL156재체(pYL156∶∶00)대침묵처리후번가화본씨연(Nicotiana benthamiana)식주적병독증상화생장영향진행분석.결과표명:pYL156∶∶00침묵처리후적번가화본씨연식주균표현출엄중적계통성병독증상,생장수도현저억제,인차,pYL156∶∶00병불시일개용우TRV유도적번가화본씨연기인침묵분석적호적대조재체；위획득경호적대조재체,시험구건료2개신적대조재체pYL156∶∶NIRi화pYL156∶∶eGFP,타문분별통과재pYL156∶∶00재체다극륭위점삽입253 bp채두아초산환원매기인내함자서렬화400 bp eGFP기인편단이획득,대저2개대조재체대침묵처리후본씨연식주적병독증상화생장정황적영향여pYL156∶∶00진행비교분석.결과표명:pYL156∶∶00침묵처리후적식주표현출교엄중적계통성병독증상,생장수도현저억제,기중26.7％식주사망；pYL156∶∶NIRi침묵처리후적식주야표현출명현적병독증상이급생장수억제현상,단여pYL156∶∶00상비,소수영향교소,지유13.3％식주사망；이pYL156∶∶eGFP침묵처리후적식주불표현명현적병독증상,식주생장야불수현저영향；병독적루량검측결과현시,3개대조재체침묵처리적식주병독증상화생장수억제정황여식주체내TRV병독적적루수평밀절상관.저사결과표명,신구건적pYL156∶∶eGFP가이작위일개우월적대조재체응용우TRV유도적기인침묵분석.
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.