珍珠龙胆石斑鱼紧密连接蛋白基因Claudin-3与Claudin-12的克隆及精氨酸干预下的肠道表达  

Cloning of tight junctions protein genes Claudin-3 and Claudin-12 and their intestinal expression under the intervention of arginine in hybrid grouper Epinephelus fuscoguttatus♀×E.lanceolatus♂

作　　者：李广 崔晓 姚春凤 陈东鸿 何凯 陈冠霖 韩煜轩 杨奇慧[1,3] 迟淑艳 LI Guang;CUI Xiao;YAO Chunfeng;CHEN Donghong;HE Kai;CHEN Guanlin;HAN Yuxuan;YANG Qihui;CHI Shuyan(College of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China;Guangdong Yuehai Feeds Group Company Limited, Zhanjiang 524000, China;Southern Marine Science and Engineering Guangdong Laboratory(Zhanjiang), Zhanjiang 524025, China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东粤海饲料集团股份有限公司,广东湛江524000 [3]南方海洋科学与工程广东省实验室(湛江),广东湛江524025

出　　处：《大连海洋大学学报》2022年第1期61-70,共10页Journal of Dalian Ocean University

基　　金：广东省自然科学基金(2017A030313155);南方海洋科学与工程广东省实验室(湛江)项目(ZJW-2019-06);广东省大学生创新创业项目(S202010566003)。

摘　　要：为评价Claudin-3与Claudin-12基因在珍珠龙胆石斑鱼Epinephelus fuscoguttatus♀×E.lanceolatus♂生长和肠道黏膜屏障中的作用,通过同源克隆及RACE PCR技术克隆珍珠龙胆石斑鱼Claudin-3和Claudin-12基因的全长,采用qRT-PCR技术分析Claudin-3和Claudin-12基因在珍珠龙胆石斑鱼脑、鳃、皮肤、肌肉、肝脏、胃、前肠、中肠、后肠、头肾、体肾、脾和心脏13种组织的表达情况,并通过饲料精氨酸干预评估上述两个基因在肠道的表达情况。结果表明:Claudin-3 cDNA全长序列为1544 bp,包括一个153 bp的5′末端非翻译区(UTR)和743 bp的3′UTR,Claudin-3的开放阅读框(ORF)为648 bp,可编码215个氨基酸的蛋白,预测该蛋白相对分子质量为23100;Claudin-12 cDNA全长序列为1236 bp,包括一个118 bp的5′UTR和92 bp的3′UTR,Claudin-12的ORF为1026 bp,可编码341个氨基酸的蛋白,预测该蛋白相对分子质量为37400;系统进化树和氨基酸序列比对显示,珍珠龙胆石斑鱼Claudin-3与斜带石斑鱼亲缘关系最近,同源性最高,一致性高达98.14%,珍珠龙胆石斑鱼Claudin-12与䲟的亲缘关系最近,同源性最高,一致性高达91.75%;Claudin-3和Claudin-12基因在被检测的13种组织中均有表达,Claudin-3 mRNA在鳃和体肾中表达量最高(P<0.05),其次是中肠和肝脏,Claudin-12在脑中表达量最高(P<0.05),其次是中肠和后肠;饲料中精氨酸含量为3.06%时,珍珠龙胆石斑鱼增重率及肠道Claudin-3和Claudin-12基因表达水平显著高于1.96%和3.74%精氨酸组(P<0.05)。研究表明,Claudin-3和Claudin-12基因表达在适宜精氨酸水平的影响下上调,可维护石斑鱼肠道黏膜屏障完整,并促进鱼体生长。To evaluate the role of Claudin-3 and Claudin-12 genes in growth and intestinal mucosal barrier of hybrid grouper Epinephelus fuscoguttatus♀×E.lanceolatus♂,the full length of Claudin-3 and Claudin-12 genes of hybrid grouper with body weight of(11.0±0.5)g were cloned by homologous cloning and the expression of Claudin-3 and Claudin-12 genes was detected in brain,gill,skin,muscle,liver,stomach,foregut,midgut,hindgut,head kidney,body kidney,spleen and heart by race qRT-PCR.The expression of these two genes in intestine and growth performance were evaluated in the hybrid grouper with initial body mass of(20.79±0.09)g fed diets containing 1.96%,3.06%and 3.74%of arginine for 8 weeks at water temperature of(29±1)℃.The results showed that the Claudin-3 had full-length cDNA sequence of 1544 bp,including a 153 bp 5′untranslated region(UTR)and a 743 bp 3′UTR,with the open reading frame(ORF)of 648 bp encoding a predicted protein of 215 amino acids with a relative molecular mass of 23100.The Claudin-12 had full-length cDNA sequence of 1236 bp,including a 118 bp 5′UTR and a 92 bp 3′UTR,with the ORF of 1026 bp encoding a predicted protein of 341 amino acids with a relative molecular mass of 37400.Phylogenetic tree and amino acid sequence alignment revealed that Claudin-3 amino acid sequence was the closest to that of grouper Epinephelus coioides with 98.14%homology,and Claudin-12 amino acid sequence was the closest to that of live sharksucker Echeneis naucratus with 91.75%homology.Claudin-12 gene and Claudin-3 gene were expressed in all 13 tissues tested,significantly higher Claudin-3 mRNA expression in gill and body kidney than those in other tissues(P<0.05),followed by midgut and liver.There was the maximal expression of Claudin-12 gene in brain,significantly higher than those in other groups(P<0.05),followed by midgut,hindgut,heart,body kidney,head kidney,and spleen.Significantly higher growth was observed in the hybrid grouper fed the diet containing 3.06%arginine,than that in the other two groups(P<0.05).

关 键 词：珍珠龙胆石斑鱼 紧密连接蛋白 基因克隆 精氨酸 

分 类 号：S917.4[农业科学—水产科学]