Comparison Of The RNA Silencing Mediated By Sequences Flanking The Inverted Repeat Construct In Different Positions

In recent years, RNA silencing is identified as a general and ancient defense mechanism. RNA silencing is conserved among various eukaryotic organisms to counter the proliferation of alien sequences, such as transposable elements, transgenes and viruses. Plant binary vector pRIR202 that containing a inverted repeat configuration was inserted with 521 bp fragment of GFP gene in the position of the upstream, middle spacer (Loop region) and the downstream of the inverted repeat respectively. By using the transient expression system, we try to evaluate RNA silencing induced by sequences flanking the different positions of inverted repeat so as to provide a theoretical basis for creating multi-virus resistant transgenic plants that can maintain the high level and long term of RNA-mediated virus resistance. The main results are as follows:1. A 521 bp fragment of GFP gene was inserted into the upstream, middle spacer (Loop region) and downstream of the inverted repeat structure of plant binary vector pRIR202 respectively, These three recombinant vectors were designated as pRIR202UP, pRIR202MID and pRIR202DWN and then transformed into the Agrobacterium tumefaciens strain C58C1.2. Plants of Nicotiana benthamiana line 16C were agroinfiltrated with pRIR202UP, pRIR202MID and pRIR202DWN respectively, the plants infiltrated with empty pRIR202 as control. The GFP fluorescence was monitored under 365 nm long-wave UV illumination and the photos were taken 4, 7, 9, 13 days after infiltration. Red ring around the infiltrated patch appeared 4 days after infiltrated with pRIR202UP, pRIR202MID, pRIR202DWN. The areas of the red ring expanded and the silencing effects enhanced as time went on. The silencing effects caused by pRIR202MID and pRIR202DWN were better than by pRIR202UP3. The values of the GFP mRNA accumulation were compared by the quantitative RT-PCR. 9 days after inoculation. N. benthamiana line 16C plants infiltrated with pRIR202 showed the high level of GFP mRNA accumulation, plants infiltrated with pRIR202UP showed 0.6598, plants infiltrated with pRIR202MID showed 0.4175, plants infiltrated with pRIR202DWN showed 0.3209. After 23 days, the results were 0.2169, 0.1996, and 0.1654. The accumulation of GFP mRNA in plant infiltrated with pRIR202UP was the highest.4. GFP accumulation level was analyzed by western blot. The results showed that GFP accumulation in plants infiltrated with pRIR202UP, pRIR202MID and pRIR202DWN were lower than with pRIR202. While the plants infiltrated with PRIR202UP showed the highest level of value.