Study Of Disease Resistant On Grass Carp Reovirus By RNA Interference(RNAi)

Gene-Modified technology was developed in 1980s which targeted to modify the genetic elements by the foreign DNA trasfered to oosperms or earlyl embryoes. In fishery, the new gene-modified oragnism (GMO) had better characters such as growth faster,disease-resistant, antifreeze and tolerance of salt or alkali . Grass carp (Ctenopharyngodon idellus) belonging to cyprinid family is a popular commercial fish species in China. Hemorrhagic disease caused by grass carp reovirus virus(GCRV) leads to more than 90% mortality and huge economic loss to the aquaculture industry. So far, there have been no effective measures to control the viral infection. Recently, a significant new strategy called RNA interference (RNAi),which mediated by small interfering RNA (siRNA) or small hairpin RNA(shRNA) belonging to post-transcrptional gene silencing(PTGS) was used to defend disease. RNAi is a gene silencing process via sequence specific destruction of mRNA in plants and animals triggered by siRNAs that shares the identical sequence with the target gene. Therefore, RNAi could target the viral message RNA to specifically silence viral protein synthesis. The shRNAs expression vector directed by H1-RNA gene promoter was trasnfered to culture cell or embryos for long-term expression. This project aims to pineer new approaches developed in the last few years to tackle the viral diseases in fish. For such purpose, we used grass carp and GCRV as a model system to study RNAi antiviral functions in fish and combined transgenic technology to potentially generate disease resistant fish strains. For such purposes, the main results were presented as followed: (1)The H1 gene and H1 promoter was cloned from grass carp: One pair of degenerate primers was designed according to the sequenced H1 RNA gene from human,mouse,zebrafish et al. The H1-RNA gene was cloned from grass carp as 301bp.Then three special primers on the grass carp H1-RNA gene sequence were designed to clone the H1-RNA promoter as 738bp by TAIL-PCR method. (2)The activity analysis of gc-H1 promoter: The three gc-H1 promoter fragments in different length named H1A(165bp),H1B(322bp),H1C(623bp) were amplified by PCR using three forward primers HP1A, HP1B, HP1C, respectively and the only reverse primer HP1.The three shRNAs expression vectors named pH1AsiGFP-CMVeGFP,pH1BsiGFP-CMVeGFP,pH1CsiGFP-CMVeGFP targeting EGFP (122-142),In which the GFP expression-suppressed cassette as pH1(x)siGFP and the GFP expression cassette as pCMVeGFP were constructed in the same vectors. The three vectors were microinjected to the embryos of zebrafish with 50 ng/μL. The mRNA level of GFP gene analyzed by real-time PCR resulted that 30%-40% mRNA of GFP were degradated, and the activity of three H1 promoter fragements was similar. The GFP protein expression levels were 50%-60% comparative to the control by statistical ratio of embryos showing green fluorescence. The ratios injected with three pH1(x)siGFP-CMVeGFP vectors were no difference, but significantly suppressed comparative to those pCMVeGFP-injection embryos (control)(**P<0.01). The results suggested that the three gc-H1 promoter fragements were long enough to trasncrapt functional shRNAs. We decided the H1B(322bp) as the only H1 promoter for the next experiments to drive the shRNAs expression.(3) Select the better types and the suitable dose of siRNA for gene silencing: Three GFP-expression-silencing vectors as the shRNA expression vector (pH1siGFP),the antisene RNA expression vector( pH1antiGFP) and the synthsis siRNA(siGFP) were co-injected in various concentrations with 50ng/μL pCMVeGFP. The concentration grads of 50-, 250-, 500-, 750 ng/μL corresponded to ratios 1:1, 5:1, 10:1, 15:1, respectively. The embryos with pH1siGFP-or siGFP-injection at the same concentration had no difference in gene silencing while the dose-dependenc was obvious. In 1:1 group, pH1siGFP-or siGFP-injection embryoes had 51.7%,42.8% embryos showing GFP, respectively. That resembled to pH1(x)siGFP-CMVeGFP on qunatity and intensity. While in 5:1 group, 30.5% embryos showed sporadical GFP. Increasing pH1siGFP to 500 ng/ μL(10:1) ,750 ng/ μL(15:1),the luminescent embryos reduced to 14.9%,5.2%,respectively. The silencing effects enhanced much singnificantly that hardly green fluorescence are visible, but the mortality of embryos also rising. The four groups with diffetent concentration of pH1siGFP were statistically significant (**P<0.01). The results with various siGFPs were similar to pH1siGFP. The results of Real-Time PCR supported the previous description. The GFP expression were suppressed about 24.5%,49.9%,72.6%,91.2% in the embroys injected with pH1siGFP of 1:1, 5:1, 10:1, 15:1 concentration, respectively. And the siGFP suppressed from 29.7% to 94.7%. The latter was a little stronger than the former, but the two had no statistically significant difference. In addition, the antisense RNA expression vector of pH1antiGFP was also co-injected to zebrafish embryos in four concentration with 50ng/μL pCMVeGFP. The luminescent embryos were 71%, 49.5%, 28%,19.1% , while the expression of GFP mRNA was suppressed to 15.7%, 35%, 52.5%, 69.1%,respectively.Therefore both the quantity and the intensity of GFP suppressed by pH1antiGFP were weaker than pH1siGFP or siGFP with statistically significance(**P<0.01). To investigate whether siRNA affects the protein expression level of target genes by reducing the level of target mRNA, this sequence-specific suppression was further confirmed byWestern blot analysis using GFP antibody. The results supported the conclusions above qualitatively. The results meant the gene silencing of siRNAs was dose-dependent, higher concentration stronger silence. But the high concentration of foreign gene was toxic, which might result in the largely death of the embryos. Therefore , the 250-500 ng/μL of siRNAs or pH1siGFP was appropriate in our experiments. (4)Screening of effective RNAi sites targeted to GCRV:Three RNAi vectors were constructed targeting GCRV virus caspid protein VP7 named pH1siGCRV1-CMVeGFP, pH1siGCRV2-CMVeGFP and pH1siGCRV3-CMVeGFP. Various RNAi constructs transfected into grass carp kidney cells(CIK cells). Such transfected cells were infectied by GCRV 991 strain. For the plaque-forming, the pH1siGCRV2-CMVeGFP had the strongest suppression for GCRV replication. (5)RNAi transgenic fish and resistance to viral infection: Two gene-modified fish were gained by microinjected the RNAi vector pH1siGCRV2-CMVeGFP and the control vector pH1siHK-CMVeGFP to Gobiocypris rarus embryoes. After 3 months, Such fish were infected with GCRV 991 strain. The fish with pH1siHK-CMVeGFP or without forgein gene were 100% dead in 12dpi (day-post-infection), while the fish with pH1siGCRV2-CMVeGFP were only 30% dead lasting to 20 dpi.The total RNA was extracted from spleen, hindgut, liver, gill, muscle on 2,5,8,11,16,23 dpi. The results of real-time PCR on Mx gene suggested that the fish infected with GCRV induced interferons, while the viral-defence was caused by RNAi. The results of real-time PCR on VP7 gene suggested that siRNAs targeted VP7 suppressed the replication of virus. The gc-H1 promoter driven shRNA strong suppressed the forgein gene expression in fish culture cells,fish embryoes and fish oragnisms. This paper firstlypresented the fish H1-RNA promoter could drive the shRNAs transcrption effectively and could silence the targeting gene in fish oragnisms.