文献类型：期刊文献

中文题名：珍珠龙胆石斑鱼闭锁小带蛋白-1、闭锁蛋白和跨膜蛋白-15a基因的克隆及其在精氨酸干预下的肠道组织表达

英文题名：Clone of Zonula Occluden-1, Occludin and Claudin -15 a Genes from HybridGrouper ( Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus) andIts Expression in Intestinal Tissue under Intervention of Arginine

作者：陈东鸿[1];崔晓[1];李广[1];何凯[1];韩煜轩[1];陈冠霖[1];杨奇慧[1,2];迟淑艳[1,2]

机构：[1]广东海洋大学水产学院,湛江524088;[2]南方海洋科学与工程广东省实验室(湛江),湛江524025

年份：2021

卷号：33

期号：8

起止页码：4645

中文期刊名：动物营养学报

外文期刊名：CHINESE JOURNAL OF ANIMAL NUTRITION

收录：CSTPCD、、CSCD2021_2022、北大核心、CSCD、北大核心2020

基金：广东省自然科学基金项目(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)。

语种：中文

中文关键词：珍珠龙胆石斑鱼;ZO-1;Occludin;Claudin-15 a;精氨酸

外文关键词：Epinephelus fuscoguttatus♀×Epinephelus lanceolatus;ZO-1;Occludin;Claudin-15a;arginine

中文摘要：本试验旨在研究珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus)闭锁小带蛋白-1(ZO-1)、闭锁蛋白(Occludin)和跨膜蛋白-15a(Claudin-15 a)基因的克隆及其在精氨酸干预下的肠道组织表达。通过cDNA末端快速扩增(RACE)-PCR技术克隆珍珠龙胆石斑鱼ZO-1、Occludin和Claudin-15 a的cDNA全长,运用实时荧光定量PCR方法分析其在健康珍珠龙胆石斑鱼脑、鳃、皮肤、肌肉、肝脏、胃、前肠、中肠、后肠、头肾、体肾、脾脏和心脏13种组织中的mRNA相对表达量。选择375尾珍珠龙胆石斑鱼,初重为(20.79±0.09)g,随机分为3组,每组5个重复,每个重复25尾鱼。各组饲料精氨酸水平分别为1.96%、3.06%和3.74%。养殖8周,测定肠道中ZO-1、Occludin和Claudin-15 a mRNA相对表达量。结果表明:1)ZO-1的cDNA全长为6355 bp,其中开放阅读框(ORF)为5106 bp,可编码1701个氨基酸,5′-非翻译区(UTR)和3′-UTR分别为105、1144 bp,无跨膜区域。Occludin的cDNA全长为2222 bp,其中ORF为1515 bp,可编码504个氨基酸,5′-UTR和3′-UTR分别为69、638 bp,有4个跨膜区域。Claudin-15 a的cDNA全长为1584 bp,其中ORF为669 bp,可编码222个氨基酸,5′-UTR和3′-UTR分别为387、528 bp,有5个跨膜区域。2)系统发育树和氨基酸序列比对结果表明,珍珠龙胆石斑鱼ZO-1与高体鰤的同源性最高(94%),Occludin与大黄鱼的同源性最高(84%),Claudin-15 a与环纹圆天竺鲷的同源性最高(92%)。3)后肠中ZO-1 mRNA相对表达量极显著高于其他组织(P<0.05),而脑、鳃、心脏、头肾、胃、肝脏、肌肉、体肾的ZO-1 mRNA相对表达量差异不显著(P>0.05);前肠、中肠中Claudin-15 a mRNA相对表达量显著高于其他各组织(P<0.05);皮肤、前肠、中肠、后肠和鳃中Occludin mRNA相对表达量显著高于其他各组织(P<0.05)。4)3.06%精氨酸水平组的增重率及ZO-1、Occludin、Clauin-15 a mRNA相对表达量显著高于1.96%和3.74%精氨酸水平组(P<0.05)。由此可见,本研究通过RACE-PCR技术克隆获得了珍珠龙胆石斑鱼ZO-1、Claudin-15 a、Occludin的cDNA序列长度。饲料适宜精氨酸水平可显著上调珍珠龙胆石斑鱼肠道ZO-1、Occludin、Claudin-15 a mRNA相对表达量,维护肠道黏膜屏障完整,促进鱼健康生长。

外文摘要：This experiment was conducted to study the clone of Zonula Occluden-1(ZO-1),Occludin and Claudin-15 a genes from hybrid grouper(Epinephelus fuscoguttatus♀×Epinephelus lanceolatus)and its expression in intestinal tissue under intervention of arginine.The full-length cDNAs of ZO-1,Occludin and Claudin-15 a of hybrid grouper were cloned by rapid amplification of cDNA ends(RACE)-PCR technology,and the mRNA relative expression levels of 13 kinds of tissues(brain,gill,skin,muscle,liver,stomach,fore-intestine,mid-intestine,hind-intestine,head kidney,body kidney,spleen and heart)of hybrid grouper were analyzed.A total of 375 hybrid grouper with similar body weight of(20.79±0.09)g were randomly divided into 3 groups with 5 replicates per group and 25 fish per replicate.The dietary arginine levels of each groups were 1.96%,3.06%and 3.74%,respectively.Feeding for 8 weeks,and the mRNA relative expression levels of ZO-1,Occludin and Claudin-15 a in intestine were measured.The results showed as follows:1)the full-length cDNA of ZO-1 consisted of 6355 bp,contained an open reading frame(ORF)of 5106 bp,encoded 1701 amino acids,contained a 105 bp 5′-untranslated region(UTR)and a 1144 bp 3′-UTR,and had no transmembrane region.The full-length cDNA of Occludin consisted of 2222 bp,contained an ORF 1515 bp,encoded 504 amino acids,contained a 69 bp 5′-UTR and a 638 bp 3′-UTR,and had 4 transmembrane regions.The full-length cDNA of Claudin-15 a consisted of 1584 bp,contained an ORF 669 bp,encoded 222 amino acids,contained a 387 bp 5′-UTR and a 528 bp 3′-UTR,and had 5 transmembrane regions.2)The phylogenetic tree analysis and amino acid sequence alignment showed that ZO-1 of hybrid grouper had the highest homology with Seriola dumerili(94%),the Occludin had the highest homology with Larimichthys crocea(84%)and the Claudin-15 a had the highest homology with Sphaeramia orbicularis(92%).3)The mRNA expression relative level of ZO-1 in hind-intestine was significantly higher than that of other tissues(P<0.05),while the mRNA expression relative level of ZO-1 in brain,gills,heart,head kidney,stomach,liver,muscle and body kidney was no significant difference(P>0.05);the mRNA expression relative level of Claudin-15 a in fore-intestine and mid-intestine was significantly higher than that of other tissues(P<0.05);the mRNA expression relative level of Occludin in skin,fore-intestine,mid-intestine,hind-intestine and gill was significantly higher than that of other tissues(P<0.05).4)The weight gain rate and mRNA expression relative levels of ZO-1,Claudin-15 a and Occludin in intestine of 3.06%arginine level group were significantly higher than those of 1.69%and 3.74%arginine level groups(P<0.05).In conclusion,the full-length cDNAs of ZO-1,Occludin and Claudin-15 a of hybrid grouper are cloned by RACE-PCR technology in this experiment.Dietary appropriate arginine level can significant improve the mRNA expression relative levels of ZO-1,Claudin-15 a and Occludin in intestine of hybrid grouper,maintain the intestinal mucosal barrier integrity,and promote the healthy growth of fish.