Expression Change Of Genes Involved In Litopenaeus Vannamei RNA Interference Under Challenge Of Six Double-stranded RNAs

To elucidate the mechanism of anviral immunity induced by double-stranded RNA(dsRNA), the expression change of Litopenaeus vannamei genes involved in RNAinterference (RNAi) under chanllenge of six dsRNAs were investigated.Through construction of recombinant vectors and in vitro transcription, six dsRNAs wereprepared, namely Rab7-dsRNA, Dicer2-dsRNA, Hemocayanin-dsRNA, CDP-dsRNA,Dicer1-dsRNA and IFNG-dsRNA. The transcription template of IFNG-dsRNA was genomicDNA fragment of Qinchuan Cattle, while the template of other five dsRNAs were all cDNAfragment of Litopenaeus vannamei. These dsRNAs were intramuscularly injected intoLitopenaeus vannamei (6~9g in body weight) obtained from the same family. Total RNA wasextracted at6h,12h,24h and48h post injection and reverse transcribed into cDNA.Expressions of18genes in those three tissues were detected by real time quantative PCR.In this study we obtained the following results:1. The up-regualtion patterns of RNAi related genes (sid-1, Dicer2and Argonaute2)were different under challenge of different sequence-specific dsRNAs. Under the challenge ofnonspecific IFNG-dsRNA, the up-regulation of RNAi related genes was concentrated on earlystage post injection, then the up-regulations falled quickly to normal level. However, theup-regulation of RNAi related genes under challenge of specific dsRNAs were prolonged.This indicate that the RNA-induced silencing complex (RISC) assembled under induction ofnonspecific dsRNA (i.e. IFNG-dsRNA) was more stable than that assembled under inductionof specific dsRNAs.2. The silencing efficiency of different genes varied greatly. Hemocyanin and CDP genescould be effectively silenced, while Dicer2and Dicer1genes could hardly be silenced. Thesilencing efficiency of Rab7gene was in the middle.3. dsRNA could cause down-regulation of untargeted genes, which is called off-targeteffects. Down-regulation of genes caused by off-terget effects was moderate compared tosequence-specific gene silencing, which was generally no more than3fold. The occurence ofoff-terget effects was dependent on sequence match. When the match is less than10nucleotides, off-target effects could hardly happen. 4. Rab7gene may act as a weak negative regulator of RNAi. Apart from Rab7-dsRNA,all other five dsRNAs also caused down-regulation of Rab7gene. Since the sequence matchbetween Rab7gene and other dsRNA sequences were all less than10nucleotides, thedown-regulation of Rab7gene caused by those dsRNAs were not likely all due to off-targeteffects.The results obtained in this study indicate that the antiviral immunity caused bynonspecific dsRNA is most probably due to the increasing amount of free Dicer2andArgonaute2proteins in cells. Although the up-regulation of RNAi related genes underchallenge of dsRNA are transient, the antiviral effect can last longer. According to thishypothesis, dsRNAs targeting shrimp endogenous genes could induce antiviral immunity,which is testified by literatures.