| Grass carp (Ctenopharyngodon idellus) is the largest output of freshwater cultured species in China currently, but its resistance of illness is low, so grass carp is ill easily, and viral hemorrhagic disease of grass carp is one of the most serious diseases, which has caused economic loss to the aquaculture industry hardly. The method using pathogen selective breeding of resistant individual to cultivate resistant family is one of important focus of the solutions. Our team have engaged in the selective breeding of grass carp anti hemorrhagic virus 2010, and have achieved sexually mature male parent (M♂) and their off-spring (F1). To verify the feasibility of the selective breeding of grass carp anti hemorrhagic virus, M♂,F1, ordinary grass carp parent (P♂) and their off-spring (f1) and barbel chub (Squaliobarbus curriculus) progeny (c1) were choosed as the research object, physiological and biochemical indexes of blood and expression of 13 immune factor closely related with the grass carp hemorrhage in different tissues were analyzed, and 4 new genes related to the grass carp hemorrhage were cloned, results are as bellow:1. indexes of blood physiological and biochemical in resistance selected grass carp parent and offspring to GCRVAantioxidant index:the enzyme activity of serum T-AOC and CAT in M♂ were significantly higher than that of P♂; and the enzyme activity of serum SOD in M♂ were higher than that of P♂,but the difference was not significant(P>0.05), which showed the ability of anti oxidative stress of M♂ was stronger than that of P♂.Significant differences of serum T-AOC and CAT also existed amongF1,f1 and c1,with the down order of c1,F1 and f1; the enzyme activity of serum SOD in c1 and F1 was also significantly higher than that of f1, which showed c1 and F1 had stronger ability of anti oxidative stress than that of f1.Blood immune index:the enzyme activity of serum SOD and lysozyme and the concentration of IgM and IFN-alpha in M♂ were higher than that of P♂, however the concentration of serum C3 and IL-1 beta in M♂ were significantly lower than that of P♂ (P< 0.01). Among F1,c1 and f1,the enzyme activity of serum SOD and lysozyme were the highest in c1, followed by F1 and f1 respectively (P< 0.01); the levels of serum C3 and IL-1 beta also had very significant difference (P< 0.01), with the down order of f1, F1 and c1; the levels of serum IgM and IFN-alpha were highest in F1 respective!y,significantly higher than that of cl and fl (P< 0.01),which showed the anti infection ability of Fl is between cl and fl.2. Expression of 13 genes related to immune of the 5 experiment groupsBy RT-PCR, expression of 13 genes related to immune of the 5 experiment groups was anlysed, tissues include liver, spleen, muscle, kidney, head kidney, intestine, gill and gonad the results indicated that:The relative expressions of C1Q mRNA of M♂ in the liver, spleen, head kidney and gills were higher than that of P♂; the relative expression of C2 mRNA of M♂ in other tissues except in liver were higher than that of P♂; the expression of C3 mRNA of M♂ in the kidney,head kidney,gills,gut,muscle and gonad were significantly higher than that of P♂(P<0.01),however which in the liver and spleen were less than that of P♂; the relative expression of coagulation factors FⅡ and FⅦ mRNA of M♂ in liver were higher than that of P♂;the relative expression of thrombolytic PLG of M♂ in all tissues were less than that of P♂;the relative expression of NGFR mRNA of M♂ in the kidney, intestine, head kidney and gill were higher than that of P♂;the relative expression of TGFβ33 mRNA of M♂ in liver, spleen and gonadal were higher than that of P♂.Above results suggested that anti GCRV performance of M♂ was stronger than that of P♂.However, the relative expression of some factor mRNA in some tissues showed the opposite case:the relative expression of DF and C5 mRNA in eight tested tissues of M♂ were less than that of P♂;C1Q mRNA expression levels in the kidney, intestine, gonads and muscle tissue of M♂ were less than that of P♂;C2 mRNA expression levels in liver of M♂ were less than that of P♂;C3 mRNA expression levels of M♂ in liver and spleen were less than that of P♂; the relative expression of FII and FVII mRNA of M♂ in other tissues except in liver were less than that of P♂;the relative expression of NGFR mRNA of M♂ in the liver, spleen, gonads and muscle were less than that of P♂;the relative expression of TGF-P3 mRNA in the kidney, intestine,muscle,head kidney and gill were less than that of P♂,which may be due to low immune response of grass carp caused by overdose virus.The FⅡ mRNA relative expression of clin other tissues except for liver,spleen and gonad were higher than that of F1 and f1,and the relative expression of other factors except for FⅡ of c1 in all tested tissues were higher than that of F1 and f1,which showed c1 had stronger resistance than F1 and f1.In the liver, the expression of coagulation factor FⅡ and FⅦ in F1 were higher than that of f1, while the fibrinolytic factor PLG expression in F1 was lower than that of f1,which indicated that anti bleeding capacity of F1 was higher than that of f1.In fish immune organ(spleen, kidney, head kidney and liver),the expression levels of most of complement factor mRNA in F1 were higher than that of f1,which showed F1 had stronger resistance than f1;however,in the three first filial generations.the relative expression of each cell factor mRNA in different tissues showed no consistent rules,especially the expression of XCR1 mRNA in all tested tissues of F1 were lower than that of f1, which was caused still need further research.Generally speaking, the anti bleeding ability and immunity of F1 were between that of c1 and f1.3. Cloning and expression analysis of genes (FⅧ, PLG, DF, XCR1)related to immune of grass carpRACE was used to clone the gene full-length cDNA of FⅧ, PLG, DF and XCR1 of grass carp, NCBI number is KF937389, KF937390, KF672346 and KF937391.Bioinformatics analysis showed that the conserved domains as in other species were all respectively contained in FVIII,PLG,DF and XCR1 gene structure of grass carp;and clustering analysis showed that FVIII,PLG,DF and XCR1 of grass carp were clustered the first to that of fish,and their evolvement were the same with other species.by RT-PCR, expression difference of FVIII, PLG, DF and XCR1 in tissues of Liver, spleen, muscle, head kidney, mid-intestine and gill of F1 and f1 in the time of 0 h,12 h,24 h,48 h,72 h,96 h,120 h,144 h,168 h after injected GCRV were detected, results showed that resistance of F1 to GCRV was higher than thay of f1. |
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