RNA Silencing Mediated Nicotiana Benthamiana Lines for Odontoglossum Ringspot Virus or Cymbidium Mosaic Virus Resistance
|School||Hangzhou Normal University|
|Keywords||Cymbidium mosaic virus Odontoglossum ringspot virus RNA silencing Transient expression Northern blot Transgenic Nicotiana benthamiana|
RNA silencing mediated antiviral research is widely adopted as an antiviral genetic engineering in recent years. Odontoglossum ringspot virus (ORSV) and Cymbidium mosaic virus (CymMV) are the two viruses with broadest horbility and severest harm in Orchidaceae. Orchidaceae is the perennial important economic crop, but the long cultivation cycle of Explants in vitro is an important bottleneck during the process of cultivating transgenic antiviral plants. In order to overcome the above difficulties, Nicotiana benthamiana was chosen as the experimental model plant, because Nicotiana benthamiana as an ideal laboratory model plant has the features of easy operation, easy cultivation, short growth cycle and high frequency in vitro regeneration.Our laboratory constructed ORSV silencing vector and CymMV silencing vector respectively:Inverted repeats (IR) of the partial ORSV and CymMV coat protein (CP) gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Based on the principle of RNA silencing, we made use of Agroinfiltration to transiently express CP hairpin RNA (hpRNA) homologous to virus and we also inoculated the virus in Nicotiana benthamiana simultaneously.Observation of plant symptoms and detect inoculated plants susceptible situation by molecular biology techniques. In order to obtain the plants that the virus resistance could inherit and keep stable, Agrobacterium-mediated transformation was carried out and preliminary study about the anti-virus of transgenic plants was done. The research results have important theoretical significance and application value. The results of this experimental study are as follows:1、Tobacco leaves were inoculated with ORSV and CymMV in this study. The results of clear symptom in the leaves and RT-PCR suggested that virus successfully infecting tobacco. Tobacco could be the host of ORSV and CymMV and provided an ideal model plant for orchid antivirus research.2. We inoculated ORSV(CymMV) silencing vector and ORSV(CymMV) in Nicotiana benthamiana’ leaves with Agroinfiltration. Observation of plant symptoms,we found plants have no symptoms or mild symptoms.The results of Northern Blot, sqPCR and Western Blot suggested the viral genome expression level of the experimental treatment group are significantly lower than the positive control group’s.Results from the current study indicated that viral silencing vector working in plants can effectively interfere with the expression of the viral genome to protect plants from virus infection.3、In order to obtain the plants that the virus resistance could inherit and keep stable, Agrobacterium-mediated transformation and tissue culture techniques were carried out and six transgenic lines containing ORSV silencing vector and four transgenic lines containing CymMV silencing vector were successfully obtained. sRNA from To transgenic plants has been detected by PAGE/Northern blot. ORSV/CymMV silencing vector can stably express inverted repeated sequences of partial CP gene and the hairpin structure formed by transcription can be cut to produce siRNA in transgenic plants. This experiment is still further study.