Effect of stem-loop proportion in hpRNA on the RNA-mediated virus resistance

Six binary vectors with 4 ∶1,2 ∶1,1 ∶1,1 ∶2,1 ∶4 and 1 ∶8 stem-loop proportion of hpRNA were constructed,respectively.The inverted repeat DNA sequence of hpRNA derived from 3'ends 50 bp of PVYN CP gene,and the spacer sequence of hpRNA derived from different part of pUC19.Recombinant binary vectors were then introduced into tobacco(NC89)plants via Agrobacterium tumefaciens-mediated transformation system.Transgenic plants transformed with different binary vectors were obtained respectively.Resistance tests indicated that hpRNA with different stem-loop proportion had different effect on inducing RNA-mediated gene silencing.When the proportion was 4 ∶1,2 ∶1 and 1 ∶1,hpRNA could effectively induce RNAi,about 60% transgenic plants were highly resistant to PVYN infection;with the length of loop increased,the proportion of resistant transgenic plants became lower;when it was 1 ∶8,the proportion of resistant transgenic plants was only 9.52%.Southern blot analysis confirmed that the foreign gene had been integrated into the tobacco genome,and there were no relationship between resistance of transgenic plants and the transgene copy numbers.Northern blot results revealed an inverse correlation between transgenic transcript accumulation and virus resistance,it demonstrated that the resistance was RNA mediated.