Synthesis double-stranded RNA on a large scale and its application in Penaeus monodon

RNA interference(RNAi)is initiated by double-stranded RNA(dsRNA)and has been used to improve our knowledge of the shrimp immune system.RNAi has also been used on a therapeutic approach for shrimp virus control.Method for dsRNA synthesis on a large scale will facilitate the application of RNAi in farmed shrimp.Kazal proteinase inhibitor(KPI) gene of shrimp Penaeus monodon was used as an example to show a large-scale preparation of long double-stranded RNA(>300 nt) in detail by a 2-step cloning method.Two commercial vectors pGEMT and pDRIVE were used to construct a dsRNA-expression system,which transformed into RNase-deficient Escherichia coli HT115(DE3).The hairpin-RNA consisted of the target forward sequence(494 bp)and a 91-base shortened version of its inverted repeat(403 bp)to introduce a loop,no more need for direct cloning of the loop segment.The hairpin-expression vector resulted in large-scale dsRNA synthesis,the yield of dsRNA was about 1 mg dsRNA/30 mL bacterial culture,and its cost was approximately one fourth of the cost of same production by using a commercial in vitro transcription kit.A test group of shrimp Penaeus monodon(8 g,12 shrimp each group)was injected intramuscularly in the fourth or fifth abdominal segment with 16 μg dsRNA to investigate the sequence specific RNAi effect on endogenous KPI mRNA expression.The NaCl injected group and GFP-dsRNA injected group were used as control.Hemolymph(200 μL)was collected from 3 shrimp at 0 h,6 h,12 h,and 24 h. RT-PCR test showed that KPI expression was completely knocked-down at 24 h.It should be possible to produce industrial-scale dsRNA production for shrimp farms by this in vivo transcription method.