Inducible Expression Of ORMV 126kDa Replicase Gene In Arabidopsis Thaliana

Viral disease is one of the most important plant diseases.Plant virus infection not only threat seriously to the structure of agricultural production,but also cause serious damage to the agricultural economy.Therefore,the prevention and control of plant virus has long been Scientists’ concern..In last two decades,researchers find that the plant itself has a defense mechanism against plant viruses and the mechanism is RNA interference.RNA interference is post-transcriptional gene silence phenomenon mediated by double stranded RNA molecules.The small RNAs in the RNA interference mechanism of plants play a central role in fighting against viruses,some of them,eg.miRNAs,come from the precursor of non protein coding RNA.These small RNAs play a negative role in the regulation of the target mRNA by the principle of complementary base pairing,thus the utilization of sRNA to fight against plant virus is an effective method in recent years.However,in order to effectively infect plants,the plant viruses must inhibit the RNAi mechanism of plants to defend themselves.Therefore,in order to facilitate the infection of plant,the viruses encode the proteins to inhibit various steps of RNA interference,and these proteins are known as gene silencing suppressor.ORMV(oilseed rape mosaic virus)is a Tobamovirus which infects Oilseed rape.The126 kD replicase of ORMV is not only a pathogenic factor but also a gene silencing suppressor.Previous research show P126 can combine 21 nt small RNAs in vitro.We have shown that ORMV infection results not only the accumulation of endogenous sizes-specific21 nt small RNAs,but also affect the expression of their target mRNA.In order to further verify the small RNA binding and enrichment function of 126 kD replicase protein,and to better understand the influence of this protein on the expression plant endogenous transcripts,we attempted to express this protein in plants.However,our previous experiments encountered difficulties in consistently over-express this protein in plants.In this study,we use the inducible expression system to conditionally express 126 kD replicase in ArabidopsisThaliana..Silwet-L77 is a nonionic surfactant which is widely used in plant transformation.We found that applying the surfactant also enhances the E.coli transformation efficiency.Meanwhile,we optimized the cultivation temperature,the cultivation density of the E.coli for competent cells preparation.We also optimized the concentration of the freezing protection agent,DMSO.Nevertheless,our results provide an optimized method as the following: Grow the E.coli at 28℃ till the culture density OD600 reaches 0.6,Prepare the competent cells,and then store these cells with 9% DMSO in deep freezer.In the end,add 15~20ppm Silwet-L77 to the transformation mixture.