【作者】 王红权；

【导师】 易自力； 肖调义；

【作者基本信息】 湖南农业大学 ， 遗传学， 2014， 博士

【摘要】 草鱼是我国目前淡水养殖产量最大的品种,但其抗病力低,易患多种疾病,其中病毒性草鱼出血病是危害其鱼种的最严重疾病之一,给其养殖业造成了巨大的经济损失。利用病原选育抗性个体,培养抗病家系成为解决此问题的重要方法之一。本研究从2010年开始进行利用草鱼出血病病毒(grass carp reovirus, GCRV)选育抗病草鱼的工作,2013年获得了性成熟的抗性选育父本(M8)及其子代(F1);为验证草鱼出血病病毒选育抗病草鱼的可行性,对抗性选育的父本及其子代、普通草鱼父本(P(?))及其子代(f1)、赤眼鳟子代(c1)的血液生理生化指标进行了分析；同时研究了与草鱼出血病紧密相关的13个免疫因子在5组试验鱼不同组织中表达;克隆并分析了与草鱼出血病紧密相关的4个新基因。主要研究结果如下1.GCRV抗性选择对草鱼父本及其子代血液生理生化指标的影响抗氧化指标分析：M(?)血清总抗氧化能力(T-AOC)和过氧化氢酶(CAT)均极显著高于P(?)；M(?)血清超氧化物歧化酶(SOD)高于P(?),但差异不显著。说明攻毒后成活下来的草鱼比普通草鱼具有更强的抗氧化应激能力。三子代间血清T-AOC和CAT也存在极显著差异,c1最高,其次分别为F1和f1；c1和F1血清SOD也极显著高于f1,说明c1和F1均具有较强的抗氧化应激能力,而f1的抗氧化应激能力较前两者弱。血液免疫指标分析：两父本间M8血清SOD和溶菌酶活以及免疫球蛋白M(IgM)、α-干扰素(IFN-α)含量均高于P(?),但M(?)血清补体3(C3)和白细胞介素-1β (IL-1β)含量极显著低于P(?)(P<0.01)。三子代间血清SOD和溶菌酶活均以c1最高,其次分别为F1和f1(P<0.01)；血清C3和IL-1β含量也存在极显著差异(P<0.01),c1最低,其次分别为F1和fl；血清IgM和IFN-α含量以F1最高,分别极显著高于f1和c1(P<0.01)。说明F1的抗感染能力介于c1和fl间。2.草鱼13个免疫因子在5组试验鱼中的差异表达采用实时荧光定量PCR (RT-PCR),检测分析了与草鱼出血病紧密相关的13个免疫因子(XCR1、CCR4、X、NGFR、TGF-β3、FⅡ、FⅦ、PLG、DF、 C1Q、C2、C3、C5)在5组试验鱼8个组织(肝脏、脾脏、肌肉、肾、头肾、肠、鳃、性腺)中的表达。结果表明：在父本中,C1Q在肝脏、脾脏、头肾以及鳃中,M(?)高于P(?)；C2的表达量除在肝脏组织中M(?)低于P(?)外,其它组织中,M(?)、均高于P(?)；C3的表达量除在肝脏及脾脏组织中M(?)低于P(?)外,其它组织中,M(?)极显著高于P8(P<0.01)；凝血因子FⅡ、FⅦ在其合成部位-肝脏中M(?)均高于P(?),溶解血栓的PLG在所有组织中M(?)低于P8；NGFR基因在肾脏、肠道、头肾及鳃中M8均高于P8；在肝脏、脾脏、性腺组织中,TGF-P3的表达量M(?)高于P(?)；以上因子在特定部位的表达情况表明：M(?)比P(?)具有更强的抗GCRV性能。然而部分因子在不同组织中的表达却表现出相反的情况,如：DF和C5在8个组织中的表达M均低于P(?),C1Q在肾脏、肠道、性腺及肌肉组织中的表达量M(?)均低于P(?),C2在肝脏组织中的表达量M(?)低于P(?),C3在肝脏、脾脏组织中的表达量M(?)低于P(?)、,凝血因子FⅡ、FⅦ除肝脏外,在其它组织中的表达量M(?)均少于P(?),NGFR在肝脏、脾脏、性腺及肌肉中的表达量M(?)低于P(?),TGF-P3在肾脏、肠道、肌肉、头肾及鳃的表达量M(?)低于P(?)。子一代13个因子的荧光定量PCR显示,c1除了FⅡ在肝、脾和性腺中的表达低于F1和f1外,其他因子在各组织中的表达均高于F1和f1,显示了其较强的抗性。在肝脏中,凝血因子FⅡ和FⅦ在F1中的表达均高于f1,而纤溶因子PLG在F1中的表达低于f1,说明F1抗出血能力高于f1。补体系统中,在鱼类主要的免疫器官脾脏、肾脏、头肾以及肝脏中,大部分补体因子在F1中的表达均高于f1,显示了F1具有较强的抗性。细胞因子互作通路中的各因子在不同组织中的表达没有表现出一致的规律,互有高低,尤其是XCR1因子在F1各组织中的表达均低于f1,其原因有待进一步探讨。总的说来,F1的抗出血能力和免疫力介于c1、f1之间。3.草鱼免疫相关基因FⅧ、PLG、DF、XCR1的克隆与表达分析本试验利用改良RACE技术,首次克隆了草鱼FⅧ、PLG、DF及XCR1基因,其NCBI基因髩5录号分别为KF937389、KF937390、KF672346和KF937391。生物信息学分析表明,草鱼FⅧ、PLG、DF及XCR1基因结构中均包含与其它物种一样的保守结构域；聚类分析表明,草鱼FⅧ、PLG、DF及XCR1基因均与鱼类首先聚类,与其它物种的聚类远近均与物种进化方向一致。采用荧光定量PCR技术检测分析了FⅧ、PLG、DF及XCR1在Fl和f1两组鱼GCRV攻毒后不同时问点(0h、12h、24h、48h、72h、96h、120h、 144h、168h)和不同组织(肝脏、鳃、中肠、头肾、脾脏和肌肉)中的表达变化。结果表明,F1对GCRV的抗性高于f1。更多还原

【Abstract】 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.更多还原