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. 2019 Aug 30;16(12):1697–1710. doi: 10.1080/15476286.2019.1656026

Figure 2.

Figure 2.

Influence of viral synonymous SNPs on host siRNA pathway. (a) The detection of siRNA in WSSV-infected shrimp. Shrimp were infected with WSSV. At different times post-infection, wsv151 siRNA and wsv226 siRNA were detected using hemocytes of WSSV-infected shrimp by Northern blotting. U6 was used as a control. M, the synthesized wsv151 siRNA or wsv226 siRNA. (b) Sequencing of wsv151 siRNA and wsv226 siRNA. The siRNAs were cloned from WSSV-infected shrimp. The box showed the mutated base. (c) Requirement of shrimp Dicer 2 for the generation of siRNA. Shrimp were co-injected with Dicer 1-siRNA or Dicer 2-siRNA and WSSV. At different time after injection, the wsv151 siRNA and wsv226 siRNA were examined with Northern blot. U6 was used as a control. (d) The influence of synonymous SNP of wsv226 mRNA on shrimp siRNA pathway. The wild-type siRNA (WT siRNA) or SNP siRNA and wild-type mRNA (WT mRNA) or SNP mRNA were incubated with the isolated shrimp Ago2 complex. At different times after incubation, the mRNA was separated by agarose gel electrophoresis. Wsv226 mRNA alone was used as a control. (e) The role of synonymous SNP of wsv151 mRNA in shrimp siRNA pathway. The WT siRNA or SNP siRNA, WT mRNA or SNP mRNA and the isolated shrimp Ago2 complex were incubated for different time. The mRNA cleavage was examined using agarose gel electrophoresis. Wsv151 mRNA alone was used as a control. (f) The sequence analysis of wild-type and SNP mRNA and siRNA of wsv151. Boxes indicated the SNPs. The seed region and extended 3’ sup (supplementary) region of siRNA were underlined with solid and dashed lines, respectively. (g) The sequence analysis of wild-type and SNP mRNA and siRNA of wsv226. (h) The impact of synonymous SNPs on RNAi in the presence of SNP mRNA, SNP siRNA, WT mRNA and WT siRNA. SNP mRNA, SNP siRNA, wild-type (WT) mRNA and WT siRNA were mixed and then incubated with the isolated shrimp Ago2 complex. At different times after incubation, the mRNA was separated by agarose gel electrophoresis. As controls, WT siRNA+WT mRNA, SNP siRNA+SNP mRNA and mRNA alone were included in the assays. The numbers represented the incubation time (min).