杂交三倍体泥鳅减数分裂相关基因Dmc1启动子甲基化分析  

Methylation Analysis of Promoter of Dmc1 Gene Related to Meiosis in Hybrid Triploid Loach Misgurnus anguillicaudatus

作　　者：马可馨 李逸帆 杨悦瑶 李川 郭文轩 庄子昕 王伟 周贺 MA Kexin;LI Yifan;YANG Yueyao;LI Chuan;GUO Wenxuan;ZHUANG Zixin;WANG Wei;ZHOU He(Key Laboratory of Mariculture&Stock Enhancement in North China's Sea,Ministry of Agriculture and Rural Affairs,Key Laboratory of Applied Biology and Aquaculture of Northern Fishes in Liaoning Province,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学重点实验室,辽宁省北方鱼类应用生物学与增养殖重点实验室,辽宁大连116023

出　　处：《水产科学》2025年第2期244-253,共10页Fisheries Science

基　　金：国家重点研发计划“蓝色粮仓科技创新重点专项”(2018YFD0900200).

摘　　要：为探究减数分裂特异重组酶1(Dmc1)在杂交三倍体泥鳅不育机制的重要作用,本试验克隆了杂交三倍体泥鳅(4n♀×2n♂)Dmc1基因,分析了Dmc1基因的理化性质和系统发育地位。用亚硫酸氢盐测序技术和逆转录PCR技术分析了泥鳅杂交三倍体子代及其亲本性腺组织中Dmc1基因启动子区甲基化发生率和表达量。结果显示,Dmc1基因全长3895bp,开放阅读框为1029bp,编码342个氨基酸,Dmc1蛋白的预测分子质量为37.85ku,理论等电点为6.95。氨基酸多序列比对表明,泥鳅Dmc1氨基酸序列与其他鲤形目鱼类Dmc1氨基酸序列相似性较高,与节肢动物的相似性较低。系统发育进化树结果显示,泥鳅与其他硬骨鱼类Dmc1聚于同一进化支。亚硫酸氢盐测序结果显示,四倍体母本、二倍体父本及三倍体子代Dmc1基因的启动子区域甲基化发生率分别为100%、80.0%及82.2%。三倍体雄性子代Dmc1启动子区甲基化发生率高于二倍体父本,Dmc1基因表达量显著低于二倍体父本;三倍体雌性子代Dmc1启动子区甲基化发生率低于四倍体母本,Dmc1基因表达量显著高于四倍体母本。根据Pearson相关系数分析可知,Dmc1基因启动子区的甲基化发生率与其表达水平呈负相关(r=-0.986,P=0.014)。综上所述,在不同倍性泥鳅中Dmc1基因的表达量受启动子区域甲基化的负调控作用,因此推测Dmc1基因的DNA甲基化发生率与三倍体泥鳅的减数分裂异常及低育性具有一定的相关性。试验结果可为解析杂交三倍体泥鳅不育的分子机制提供理论依据。In order to investigate the important role of DNA Meiotic Recombinase 1(Dmc1)in the infertility mechanism of the hybrid triploid loach Misgurnus anguillicaudatus(4n♀×2n♂),the Dmc1 gene was cloned from the hybrid triploid loach,and the physicochemical properties and phylogenetic position of the gene were analyzed by bioinformatics.The methylation and expression levels of the promoter region of Dmc1 gene were analyzed by bisulfite sequencing(BSP)and RT-PCR in the gonadal tissues of female and male offspring of loach triploid crosses and their parents.The results showed that the Dmc1 gene had the full length of 3895 bp,and an open reading frame(ORF)of 1029 bp encoding 342 amino acids,with the predicted molecular weight of 37.85 ku,and the theoretical isoelectric point(pI)of 6.95.Multiple sequence alignment showed that the amino acid sequence of Dmc1 in the loach was similar to that of other fish in Cypriniformes and less similar to arthropods.The phylogenetic tree revealed that the loach clustered with other osteichthyans in terms of Dmc1 evolution.BSP analysis showed that the methylation rates of the Dmc1 promoter region were 100%in the tetraploid maternal parent,80.0%in diploid paternal parent,and 82.2%in triploid offsprings.There was higher methylation rate of the Dmc1 promoter region in the male offspring of triploids than that in the diploid paternal parent,with the significantly lower expression level of the Dmc1 gene than that in the diploid paternal parent.The methylation rate of the Dmc1 promoter region was shown to be lower in the female offspring of triploids than that in the tetraploid maternal parent,with significantly higher expression level of the Dmc1 than that in the tetraploid maternal parent.Pearson correlation coefficient analysis indicated that the methylation rate of the Dmc1 promoter region was negatively correlated with its expression level(r=-0.986,P=0.014).These findings suggested that the expression level of the Dmc1 gene in different ploidy loach was negatively regulated by methylati

关 键 词：泥鳅 杂交三倍体 Dmc1 启动子甲基化 

分 类 号：S917[农业科学—水产科学]