Studies On The Inhibition Of Replication And Multiplication Of BmNPV Using RNAi Technology

To exploit the inhibition effect of RNAi on the replication and multiplication of Bombyx mori Nuclear Polyhedrosis Virus (BmNPV), we selected the immediate early gene 1 (ie-I), helicase and gp64 as the target genes, which play important roles in the replication and the horizontal transmission of BmNPV DNA.The corresponding dsRNAs were designed and synthesized in vitro and were transfected with BmNPV DNA into BmN cells by lipofectin. The inhibition effects of the tested dsRNAs on the replication and multiplication of BmNPV were investigated. Through comparison of the inhibition effects of different length dsRNAs and co-inhibition of two target genes on the virus titer, the ideal target gene sequence was achieved. The investigation results maybe as references on selecting the target genes against BmNPV in the field of breeding silkworm variety using transgenic and RNAi technology methods in the future.1. The inhibition effects of the corresponding dsRNA of ie-l on the replication and multiplication of BmNPVAccording to the gene sequence of ie-l in the BmNPV genome, the 438 bp long dsRNA was designed and synthesized in vitro and was transfected with BmNPV DNA into BmN cells by lipofectin. The results showed that the virus replication was inhibited effectively. The maximum inhibition effect was that the viral titer decreased about 10⁴ times, compared with the control. The effective work duration for 1 μg dsRNA transfected with BmNPV DNA in BmN cells was about 2 days.2. The inhibition effects of the corresponding dsRNA of gene gp64 on the replication and multiplication of BmNPVBased on the head, middle,and hind sequences in the ORF of BmNPV gp64, three corresponding dsRNA, Gl(459bp), G2(508bp), and G3(337bp), were designed and synthesized in vitro, respectively. Furthermore, dsRNA G3 was subdivided into three shorter dsRNA fragments,G3-l(122bp),G3-2(105bp), and G3-3(109bp). The dsRNAs were transfected into BmN cells with BmNPV DNA. The results showed that all of the dsRNAs inhibited the replication of viral DNA effectively. The inhibition effects of G1, G2 and G3 were similar. The maximum inhibition effect was that the viral titer decreased about 10^3.5 times, compared with the control. The inhibition effects of G3-l,G3-2, and G3-3 were equal to or higher than that of G3.The most effective inhibition stages for the ie-1 dsRNA were at 24 hour post transfection (hpt) and 96 hpt. The curve of the inhibition effect on the viral titer was coordinate with that the deduced expression pattern of gp64 is both at early stage and at late stage.3. The inhibition effects of the corresponding dsRNA of helicase on the replication and multiplication of BmNPVBased on the sequence of helicase, the 427bp long dsRNA was designed and synthesized in vitro and was transfected with BmNPV DNA into BmN cells by lipofectin. The results showed that the dsRNA inhibited the replication and multiplication of BmNPV effectively. The maximum inhibitipn effect was that the viral titer decreased about 10^2.92 times, compared with the control.4.Comparisons of the inhibition effects of dsRNAs targeted different genes on the replication andmultiplication of BmNPVTo investigate the inhibition effects of dsRNAs targeted different genes on the multiplication of BmNPV, the corresponding dsRNA of ie-1(11), gp64(G3), helicase(H\) and the combination of II and HI were transfected into BmN cells, respectively. The results indicated that the inhibition effect were I1>G3>H1, suggesting that the ie-l is the ideal target gene for inhibition of the replication and multiplication of BmNPV.The inhibition effects of the combination of II and HI was between those of sole treatments, indicating that the synergia phenomenon doesn't exist for them.