Study On RNAi-mediated Virus Resisitance To LSV

Lily symptomless virus(LSV),a species of the genus Carlavirus,is the most prevalent virus infecting lily plants throughout the world,which threatens the yield and commercial production of lily.A new powerful type of resistance,based upon the presence of dsRNA, known as RNA-mediated virus resistance,was characterized by a high level of resistance. RNA interference(RNAi)is a phenomenon in which double-strand RNA(dsRNA) specifically suppresses the expression of target prote in by degrading the target mRNA.,Once it was established,it could be maintained throughout the life-span of plant.Therefore,we envisaged the use of dsRNA to target the open reading frame(ORF)of 32kDa coat protein which could be an effective way to counter LSV.A fragment of 876 bp of Lily symptomless virus(LSV)CP gene cDNA sequence was amplified by reverse transcription polymerase chain reaction(RT-PCR)from the total RNA of infected lily leaves and sequenced and then analysed with biological software to look for the 470 bp conservative fragment.Using Gateway technology,the entry clone vector(pDONR201)with 876 bp LSV CP gene or its 470 bp Conservative fragment was constructed through the way of BP cloning and the RNA interference(RNAi)transformation vectors(pH7GWIWG2(â…¡))were constructed through the way of LR cloning.The RNAi vectors were obtained.The study on the resistance of RNAi vectors by Agrobacterium-mediated transient expression system was done and then according the result,analysed the resistance of different lengths of dsRNA for RNAi vector.The RNAi vectors could be transformed into Lily by Agrobacterium tumefaciens,and then it can transcribe into double-stranded RNA with ring structure to counter LSV.Lily of anti-LSV was obtained.Our results showed:(1) LSV ORF5 encoding CP which is 876 bp were obtained and consists of 32 kDa(291 aa) protein,the pI is 7.02.The BLAST search result showed that the sequence presented a very high match with the LSV CP genes in the GenBanK and its homology was 95%(2) RNAi vectors with inverted repeats of different length LSV CP gene were obtained through Gateway technology.(3) Transiently expressed RNAi vectors could specifically interfere with LSV infection.(4) 15mg/L hygromycin concentration can restrain regeneration of material.(5) Cultivated in the absence of Hyg first had effects on the transformation efficiency. (6) The result of PCR show that we have get the transgenic lily.