文献类型：期刊文献

中文题名：军曹鱼NHE3和NKAα1a基因克隆、表达模式及相关miRNA的靶向调控

英文题名：Cloning and expression analysis of NHE3 and NKAα1a in cobia(Rachycentron canadum)and targeted regulation of related miRNAs

作者：王忠良[1];刘付柏[1];黄宝松[2];李桂英[1];王菁[1];王蓓[1];陈宗发[1]

机构：[1]广东海洋大学水产学院,广东湛江524088;[2]广东省中山市三角镇农业服务中心,广东中山528445

年份：2023

卷号：47

期号：5

起止页码：39

中文期刊名：水产学报

外文期刊名：Journal of Fisheries of China

收录：CSTPCD、、Scopus、CSCD2023_2024、北大核心、CSCD、北大核心2020

基金：国家级大学生创新创业训练计划项目(CXXL2020002);广东海洋大学本科生创新团队项目(CXTD2021001);广东海洋大学2019年“冲一流”省财政专项资金建设项目;广东省普通高校创新团队项目(2021KCXTD026,2022KCXTD013)。

语种：中文

中文关键词：军曹鱼;盐度适应;cDNA克隆;荧光定量PCR;双荧光素酶

外文关键词：Rachycentron canadum;salinity adaptation;cDNA clone;quantitative real-time PCR(qPCR);doubleluciferase

中文摘要：为了解军曹鱼NHE3与NKAα1a的序列特征、盐度胁迫下的基因表达模式以及相关miRNA的靶向调控作用,实验应用cDNA末端快速扩增技术克隆了NHE3与NKAα1a的全长cDNA序列;应用实时定量PCR(qPCR)技术分析了NHE3和NKAα1a的组织特异性和盐度适应性表达模式;采用双荧光素酶报告实验检测了NHE3和NKAα1a与相关miRNA的靶向调控关系。结果显示,军曹鱼NHE3与NKAα1a基因的开放阅读框长度分别为2718和3075 bp,分别编码905和1024个氨基酸;NHE3、NKAα1a在鳃、肠、心脏等9种组织中均有表达,其中以鳃组织中的表达丰度最高。随着盐度升高,NHE3在鳃中表达量下降,低盐和高盐适应后均呈显著差异。NKAα1a基因表达量随着盐度升高出现不同趋势,在鳃和肠中受低盐和高盐影响后均显著上调,而高盐环境下其在体肾中的表达水平显著下调。在不同盐度适应过程中,NHE3、NKAα1a均以鳃组织中的表达水平最高。NHE3-pmir-GLO-WT与miR-1335-3p共转染时,较对照组相对荧光素酶活性下降,并存在极显著差异;NKAα1a-pmirGLO-WT分别与miR-1788-3p和mimic NC(对照组)的共转染结果与上述结果类似。研究表明,miR-1335-3p和miR-1788-3p可分别与NHE3和NKAα1a 3′-UTR序列结合,并下调其mRNA表达水平。NHE3和NKAα1a在物种间高度保守;NHE3主要参与低盐适应,而NKAα1a在低盐和高盐适应中均发挥作用;miR-1335-3p和miR-1788-3p可分别负向调控其靶基因NHE3与NKAα1a,从而参与军曹鱼渗透压调节。以上研究结果为进一步研究军曹鱼miRNA-mRNA渗透压调控网络提供了理论基础。

外文摘要：To study the sequence characteristics,the gene expression patterns under salinity stress and the targeted regulation of related miRNAs of NHE3 and NKAα1a in cobia,Rachycentron canadum,the full-length cDNA sequences of NHE3 and NKAα1a were cloned by rapid amplification of cDNA ends;the tissue-specific and salinity-adaptive expression patterns of NHE3 and NKAα1a were analyzed by real-time quantitative PCR;the double-luciferase reporter assay was used to detect the targeted regulatory relationship between NHE3 and NKAα1a and related miRNAs.The open reading frames of cobia NHE3 and NKAα1a were 2718 bp and 3075 bp in length,encoding 905 and 1024 amino acids,respectively.NHE3 and NKAα1a were expressed in all detected tissues,including gill,intestine,and heart,among which the highest expression abundance was found in gill.With the increase of salinity,the expression of NHE3 in gill decreased gradually,and there were significant differences between low-salt and high-salt adaptation.With the increase of salinity,the expression level of NKAα1a showed different trends,and it was significantly up-regulated in gill and intestine after being challenged by low-salt and high-salt conditions,while its expression level was significantly down-regulated in kidney in high-salt environment.In different salinity adaptation processes,the highest expression levels of NHE3 and NKAα1a were all found in the gill.When NHE3-pmirGLO-WT was co-transfected with miR-1335-3p,the relative luciferase activity decreased compared with the control group,and there was a very significant difference,and the similar results were found when NKAα1a-pmirGLO-WT was co-transfected with miR-1788-3p and mimic NC(control).The results of the double-luciferase reporter assay suggested that miR-1335-3p and miR-1788-3p could bind to NHE3 and NKAα1a 3′-UTR sequences,respectively,and downregulate their mRNA expression levels;NHE3 and NKAα1a were highly conserved among species;NHE3 was mainly involved in low-salt adaptation,while NKAα1a played a role in both low-salt and high-salt adaptation;miR-1335-3p and miR-1788-3p could negatively regulate their target genes NHE3 and NKAα1a,respectively,and thus participate in osmotic pressure regulation in R.canadum.The above results provided a theoretical basis for the further studies of the miRNA-mRNA osmotic pressure regulatory network in R.canadum.