团头鲂C3和C9基因克隆及在饥饿胁迫下的表达分析  

Cloning of Complement9 Gene and Complement3 Gene of Megalobrama amblycephala and Analysis of Its Expression under Starvation Stress

作　　者：储辛伊 孙盛明[2] 苏艳莉 蒋高中[1,2] 朱健[2] 王燚炜 Chu Xinyi;Sun Shengming;Su Yanli;Jiang Gaozhong;Zhu Jian;Wang Yiwei(Wuxi Fishery College,Nanjing Agricultural University,Wuxi,214081;Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi,214081;Shanghai Ocean University,Shanghai,201306)

机构地区：[1]南京农业大学无锡渔业学院,无锡214081 [2]中国水产科学研究院淡水渔业研究中心,农业部种质淡水渔业与种质资源利用重点实验室,无锡214081 [3]上海海洋大学,上海201306

出　　处：《基因组学与应用生物学》2020年第10期4500-4514,共15页Genomics and Applied Biology

基　　金：国家大宗淡水鱼类产业技术体系饲料原料开发与质量安全岗位(CARS-45);国家“十二五”科技支撑计划(2012BAD25B07);江苏省自然科学基金面上项目(BK20151103)共同资助。

摘　　要：补体(Complement)是存在于动物体液中及细胞表面,经活化后可介导免疫和炎症反应的蛋白。本研究采用RACE技术首次从团头鲂(Megalobrama amblycephala)cDNA文库中克隆出C3和C9基因cDNA全长序列(MaC3和MaC9),并运用qRT-PCR技术检测了MaC3和MaC9基因在团头鲂不同组织中的表达情况和饥饿胁迫后在肝脏中的时序表达模式。结果显示,MaC3和MaC9基因序列全长分别为5378 bp和2522 bp,5'UTR长为316 bp和8 bp,3'UTR长为133 bp和480 bp,开放阅读框为4929 bp和2034 bp,分别编码1642和677个氨基酸。氨基酸序列同源性分析表明,MaC3和MaC9分别与鲫鱼(Carassius auratus)补体C3和草鱼(Ctenopharyngodon idella)补体C9相似度较高,分别达78%和84%。实时荧光定量结果表明,MaC3和MaC9在脑、肝脏、脾脏、头肾、中肾、心脏、前肠、中肠、后肠、鳃、肌肉组织中均有表达且在肝脏中表达量最高,分别在肾脏和心脏中表达量最低。在实验周期21 d和28 d,持续饥饿组团头鲂肝脏中补体C3和C9基因的表达量显著低于对照组(p<0.05),但饥饿过量投喂组团头鲂C3和C9基因表达量显著高于对照组(p<0.05),饥饿再投喂组的C3和C9基因表达量与对照组差异不显著。持续饥饿组的团头鲂肝细胞中细胞核模糊变形并且肝血窦扩张,有明显的炎症反应。研究结果表明,长期饥饿胁迫下团头鲂肝损伤且补体合成能力下降,导致团头鲂机体免疫应答受到抑制作用,本研究为团头鲂免疫防御系统的研究提供了重要资料。Complement,as a kind of protein,is activated to mediate immunity and inflammation in animal body fluids and on the cell surface.This research cloned the full-length cDNAs of C3 and C9 genes(MaC3 and MaC9)from the cDNA library of Megalobrama amblycephala for the first time by using RACE technology.Also,this research tested the expression of MaC3 and MaC9 in different tissues and the expression of them in the liver following hunger stimulation by aRT-PCR.Results showed that the total length of MaC3 and MaC9 cDNA were 5378 bp and 2522 bp,including 5'UTR of 316 bp and 8 bp,3'UTR of 133 bp and 480 bp and open reading frame(ORF)of 4929 bp and 2034 bp which encoded 1642 amino acids and 677 amino acids respectively.Homology analysis indicated that the amino acid sequence of MaC3 and MaC9 had the highest similarity with those of Carassius auratus C3 and Ctenopharyngodon idella C9,at 78%and 84%,respectively.qRT-PCR results revealed that MaC3 and MaC9 were expressed in brain,liver,spleen,head kidney,middle kidney,heart,foregut,midgut,hindgut,sputum and muscle and all expressed with the highest proportion in liver.The lowest expression was found in middle kidney and heart respectively.During the experimental period 21 d and 28 d,the expression of MaC3 and MaC9 in the starvation group was significantly lower than those of the control group(p<0.05),but the expression of MaC3 and MaC9 in the overfeeding group were significantly higher than those of the control group(p<0.05).The expression of MaC3 and MaC9 in the refeeding group were not significantly different from those of the control group.Hepatocytes observation showed that displayed nucleus indistinguishable and hepatic sinusoidal expansion,which means there is a significant inflammatory response to Megalobrama amblycephala of the starvation group.This study shows the liver injury and the complement synthesis ability decreased,which led to the inhibition of the immune response of Megalobrama amblycephala under the long-term starvation.This study provides important informati

关 键 词：团头鲂 饥饿胁迫 补体 基因克隆 

分 类 号：S917.4[农业科学—水产科学]