仿刺参miR-10与arhgef3序列分析及盐度胁迫下的表达模式  

Sequence Analysis and Expression Patterns of miR-10 and Target Gene arhgef3 in Sea Cucumber Apostichopus japonicus Exposed to Salinity Stress

作　　者：勾宇晴 魏馨 马南行 刘丹 高思琪 田燚 GOU Yuqing;WEI Xin;MA Nanxing;LIU Dan;GAO Siqi;TIAN Yi(Key Laboratory of Mariculture&Stock Enhancement in North China′s Sea,Ministry of Agriculture and Rural Affairs,College of Fisheries and Life Science,Dalian Ocean University,Dalian 116023,China)

机构地区：[1]大连海洋大学水产与生命学院,农业农村部北方海水增养殖重点实验室,辽宁大连116023

出　　处：《水产科学》2024年第6期853-864,共12页Fisheries Science

基　　金：国家重点研发计划项目(2019YFD0900505)。

摘　　要：为探究盐度胁迫下仿刺参的响应过程,从转录组和miRNA的高通量测序结果中筛选差异表达的miR-10及对应的靶基因arhgef3,分析miR-10及靶基因arhgef3在低盐胁迫下的表达情况。分析miR-10的前体序列和成熟序列显示,miR-10能形成稳定的茎环结构,高度保守区域在29~51区间。靶基因arhgef3的开放阅读框长度为1728 bp,编码575个氨基酸,在第280~500氨基酸之间存在1个功能结构域。靶基因arhgef3的表达存在明显的组织特异性,在肠道、呼吸树和体腔细胞中表达显著高于其他组织(P<0.05)。低盐条件下,miR-10在24 h的表达量显著低于对照组0 h,在48 h时显著高于对照组0 h;在胁迫6 h组arhgef3基因的表达量显著高于对照组0 h。在体腔细胞转染miR-10抑制剂(inhibitor),试验组在24 h显著低于0 h,48 h显著高于0 h;转染miR-10模拟物(mimics)后试验组在6 h和48 h均显著高于0 h。在仿刺参体内转染miR-10抑制剂(antagomir)后的试验组arhgef3基因的表达量变化不明显,而转染miR-10模拟物(agomir)后试验组arhgef3基因的表达量在24 h内有较显著的提升。试验结果表明,arhgef3可以被盐度诱导表达,且说明盐度胁迫的响应过程中需要靶基因维持高的表达水平来适应盐度变化,更好地适应仿刺参的低盐响应过程。In order to investigate the response process of sea cucumber Apostichopus japonicus exposed to salinity stress,differentially expressed miR-10 and the corresponding target gene arhgef3 were screened from the transcriptome and high-throughput sequencing results of miRNAs to analyze the expression of miR-10 and target gene arhgef3 in different tissues of sea cucumber with body weight of(18.55±3.40)g exposed to a salinity of 18 for 0 h,6 h,24 h and 48 h with control group at 30 normal salinity to provide a basis for their roles in the salinity response mechanism of the sea cucumber.Analysis of the precursor and mature sequences of miR-10 showed that miR-10 formed a stable stem-loop structure with highly conserved regions in the interval between 29 and 51.The target gene arhgef3 had an open reading frame of 1728 bp encoding 575 amino acids and a functional domain between amino acids 280—500.There was significantly higher expression level of arhgef3 in intestine,respiratory tree and coelomocytes than that in other tissues(P<0.05).Under low salt conditions,the expression level of miR-10 was significantly lower at 24 h and significantly higher at 48 h than at 0 h in the control group,and the expression level of arhgef3 was significantly higher at 6 h of stress than at 0 h in the control group.Transfection of miR-10 inhibitors into coelomocytes was found to be significantly decreased at 24 h and increased at 48 h compared with 0 h in the experimental group,with significantly higher in the experimental group at 0 h at both 6 h and 48 h after transfection with miR-10 mimics than that at 0 h.The expression level of arhgef3 in the experimental group transfected with miR-10 mimic was found to be increased significantly at 24 hours,without significant change in expression level of arhgef3 in the experimental group transfected with miR-10 inhibitor in sea cucumber.The findings indicated that arhgef3 was induced by salinity,and that the target gene was required to maintain a high expression level to adapt to salinity changes

关 键 词：仿刺参 盐度胁迫 arhgef3 miRNA-10 

分 类 号：Q786[生物学—分子生物学]