| Grass carp (Ctenopharyngodon idella) is considered an economically importantaquaculture species in China with the largest production among the "Great Four CulturedFish". But there are many pathogens for grass carps, and the disease caused by GCRV (Grasscarp reovirus) results in serious losses to the aquaculture industry. The disease is widelyspread and highly lethal, leading to economic loss of several billion per year in domestic area.So far there is no highly effective drug or treatment to this virus. And the specific immunesystem of grass carp is deficient, which result in the low efficiency of vaccine. Thus,identifying critical genes in the innate immunity networks will become the basis fordisease-resistant breeding in the future.Mx (Myxovirus-resistant) is an important downstream effector molecule of interferon(IFN), which can be specifically activated by IFN. Mx is a kind of GTPase, which is typicallycharacterized by a big N terminal GTPase domain, a specific sequence of dynaminesuperfamily domain and a C terminal Leu zipper domain, so Mx protein can form a tripolymer.There are many common characteristics between mammal and fish Mx genes, but still somedifferences exist. Study on Mx protein of fish and its molecular mechanism will facilitatefurther understanding on innate immune system of fish and can be used in disease preventionof fish cultivation.In this study, to amplify the first fragment of grass carp Mx gene, degenerate PCRprimers were designed from crucian carp (Carassius auratus) ESTs. Combining the rapidamplifications of cDNA ends (RACE), the semi-quantitative PCR (RT-PCR), the real-timefluorescence quantitative PCR (qRT-PCR), expression vectors construction, plasmidtransformation, stable cell lines establishment and the bioinformatics analysis technique, wegot two Mx isoforms of grass carp (CiMx1and CiMx3) and compared the phylogeneticrelationships and anti-GCRV roles in the innate immunity of the three grass carp Mx genesincluding CiMx1(this study), CiMx2(NCBI) and CiMx3(this study). Results are as follows:The full length of CiMx1cDNA sequence is2881bp with the longest open readingframe (ORF) of1908bp encoding a peptide of635amino acids. The full length of CiMx3 cDNA sequence is3002bp with the longest open reading frame (ORF) of1893bp encoding apeptide of630amino acids. Among the15tissues including blood, brain, eye, foregut, midgut,hindgut, gas bladder, gill, head kidney, trunk kidney, heart, liver, muscle, skin and spleen ofhealthy grass carp, the three Mx genes were highest expressed in gill and head-kidney. Theygot lower expression in blood, brain, foregut, midgut, hindgut, gas bladder, trunk kidney,heart, liver and spleen. They are weak expressed in eye and muscle, and not expressed in skin.The expression of the three grass carp Mx genes was belowed the normal level at2h,8h,24h, which increased at48h time point and significantly up-regulated at72h (P<0.05) afterGCRV infection (P<0.05) in CIK cells. Whereas the expression of CiIFN-I increased at24htime point and decreased at48h time point, which then significantly down-regulated at72hpost-infection (P<0.05). The three grass carp Mx isoforms and CiIFN-I mRNA expression inCIK was significantly up-regulated after5μg/ml poly(I:C) disposition (P<0.05), which wasslowly up-regulated by5μg/ml poly(I:C) embeded stimulation (P<0.05). On the whole, theexpression of CiMx1, CiMx2, CiMx3and CiIFN-I in three tissues (spleen, headkidney andgill) was highy up-regulated compared to the control level at12h,24h,48h,72hpost-injection of GCRV (P<0.05). In addition, at all time points, the general expression levelof CiMx1was higher than the CiMx2and CiMx3. After four plasmids were transfectedrespectively into the CIK cell, the empty pCMV-EGFP-CMV vector did not influenceCiIFN-I expression almostly and the expression of CiIFN-I was up-regulated when infectedwith GCRV, but the high expression of the three Mx genes inhibit the expression of CiIFN-Ito different degrees. The antiviral assay in a96-well plate implied that the descending orderfor resistance to GCRV is CiMx1, CiMx3and CiMx2, which means CiMx2is weaker thanthe other two Mx genes.These data indicates that the expression of CiMx1, CiMx and CiMx3is not only relatedto the antiviral innate immune response, but also able to inhibit the expression of CiIFN-I in acertain extent. All these foundings will be conducive to further explore the mechanism ofhemorrhage of grass carp and provide certain theoretical and practical evidence for thedevelopment of effective measures against viral infection in aquaculture. |
