暗纹东方鲀HTR4基因的分子特征及其生长相关SNP位点筛查  

Molecular characterization of HTR4 gene and screening of growth-related SNP loci in Takifugu fasciatus

作　　者：张雯雯 喻伟峰 张宇飞 许伟鹤 张鑫宇 王涛 尹绍武 张凯 ZHANG Wenwen;YU Weifeng;ZHANG Yufei;XU Weihe;ZHANG Xinyu;WANG Tao;YIN Shaowu;ZHANG Kai(School of Marine Science and Engineering,Nanjing Normal University,Nanjing,Jiangsu 210023,China;Engineering Research Center of Specialty Aquatic Breeding and Green Efficient Farming Technology,Nanjing,Jiangsu 210023,China)

机构地区：[1]南京师范大学海洋科学与工程学院,江苏南京210023 [2]江苏省特色水产育种与绿色高效养殖技术工程研究中心,江苏南京210023

出　　处：《西北农林科技大学学报（自然科学版）》2023年第12期18-28,共11页Journal of Northwest A&F University(Natural Science Edition)

基　　金：江苏省自然科学基金青年基金项目(BK20210563);国家自然科学基金项目(32172948);江苏省种业振兴“揭榜挂帅”项目(JBGS[2021]034)。

摘　　要：【目的】明确暗纹东方鲀(Takifugu fasciatus)5-羟色胺受体4(5-hydroxytryptamine receptor 4,HTR4)的分子结构、表达特征和功能,并探究HTR4基因SNP位点与暗纹东方鲀生长的相关性。【方法】采用分段克隆方法,分别扩增HTR4基因的3′RACE、5′RACE以及中间保守区域,将其拼接后获得暗纹东方鲀HTR4基因cDNA全长序列,对其进行生物信息学分析;利用实时荧光定量PCR技术,分析HTR4基因在暗纹东方鲀脑、肾脏、肝脏、脾脏、心脏、卵巢、中肠、鳃、皮肤和肌肉等10种组织中的分布,以及其在卵巢5个不同发育时期(Ⅰ~Ⅴ期)的脑、肾脏、肝脏、脾脏、心脏、中肠、鳃和肌肉等8种组织中的表达模式;利用Sanger测序法,筛查HTR4编码区生长性状相关的SNP位点。【结果】暗纹东方鲀HTR4 cDNA全长2302 bp,开放阅读框长度为1170 bp,共编码389个氨基酸,包含5个外显子;氨基酸序列比对及系统进化树分析结果显示,暗纹东方鲀与红鳍东方鲀HTR4的一致性最高(99.53%),亲缘关系最近;三维结构预测显示,HTR4蛋白在鱼类中较为保守。荧光定量PCR结果表明,HTR4基因在暗纹东方鲀10个不同组织中均有表达,其中在脑组织表达量最高,其次是中肠,肌肉组织表达量最低;HTR4基因在卵巢5个不同发育时期的8个不同组织中均有表达,其中在卵巢发育的Ⅳ时期表达量最高。SNP位点筛查结果表明,暗纹东方鲀HTR4基因第4外显子1202 bp处存在1个同义突变SNP位点(c.1202 C>G),关联分析表明该位点与暗纹东方鲀肥满度性状显著相关(P<0.05),其中CC基因型个体肥满度较高。【结论】HTR4基因在暗纹东方鲀生长发育中发挥了重要作用,可作为优良候选基因,在一定程度上可为暗纹东方鲀生长性状新品种的选育提供分子标记。【Objective】This study investigated molecular structure,expression characteristics and function of the HTR4 gene in Takifugu fasciatus and explored the correlation between HTR4 gene and puffer growth.【Method】The 3'UTR,5'UTR and intermediate fragments of HTR4 gene were separately amplified by segmental cloning,the full-length cDNA sequence was obtained after splicing,and bioinformatics analysis was performed.The expression pattern of the HTR4 gene in 10 tissues including brain,kidney,liver,spleen,heart,ovary,midgut,gill,skin and muscle and 8 tissues of ovary at 5 different developmental stages(Ⅰ-Ⅴ)including brain,kidney,liver,spleen,heart,midgut,gill and muscle was analyzed by quantitative real-time PCR technology.Finally,Sanger sequencing was used to screen the SNP loci related to growth traits in the coding region of HTR4.【Result】The cDNA full-length sequence of HTR4 gene in largemouth bass was 2302 bp in length with a 1170 bp open reading frame encoding 389 amino acids and 5 exons.The amino acid sequence alignment and phylogenetic tree analysis showed that T.fasciatus had the highest identity(99.53%)with Takifugu rubripes.Spatial structure of HTR4 protein showed that it was conserved in fish.The HTR4 gene was expressed in 10 different tissues.The highest expression level was in brain,followed by midgut,and the lowest expression was in muscle tissue.The HTR4 gene was expressed in 8 different tissues of the ovary at five different developmental periods,with the highest expression in theⅣperiod of ovarian development.A synonymous mutant SNP locus(c.1202 C>G)at 1202 bp in exon 4 of the HTR4 gene of T.fasciatus was screened and association analysis showed that the SNP loci had significant correlation(P<0.05)with fatness in T.fasciatus.Individuals with CC genotype had higher fatness than others.【Conclusion】The HTR4 gene played important roles in the growth and development of T.fasciatus.It can be used as a candidate gene and molecular maker for selection of new varieties.

关 键 词：暗纹东方鲀 5-羟色胺受体4 基因克隆 单核苷酸多态性 动物育种 

分 类 号：S965.399[农业科学—水产养殖]