栉孔扇贝miR-124-3p_4靶基因的筛选及dpgn-like基因的表达特征  

Target genes identification of miR-124-3p_4 and expression characteristics of dpgn-like gene in Chlamys farreri

作　　者：陈越 张瑞琦 李茜茜 黄晓婷[1] 张志峰[1,2] 秦贞奎[1] CHEN Yue;ZHANG Ruiqi;LI Xixi;HUANG Xiaoting;ZHANG Zhifeng;QIN Zhenkui(Ministry of Education Key Laboratory of Marine Genetics and Breeding,Ocean University of China,Qingdao 266003,China;Key Laboratory of Tropical Aquatic Germplasm of Hainan Province,Sanya Oceanographic Institution,Ocean University of China,Sanya 572000,China)

机构地区：[1]中国海洋大学,海洋生物遗传学与育种教育部重点实验室,山东青岛266003 [2]中国海洋大学三亚海洋研究院,海南省热带水产种质重点实验室,海南三亚572000

出　　处：《中国水产科学》2024年第6期627-639,共13页Journal of Fishery Sciences of China

基　　金：国家重点研发计划项目(2018YFD0901400)。

摘　　要：为探究微小RNA(miRNA)在贝类性腺发育过程中发挥的作用,以栉孔扇贝(Chlamys farreri)为研究材料,以在幼贝早期性别分化阶段精巢中高表达的miR-124-3p_4为研究对象开展研究,预测并验证了其靶向调控的基因,揭示关键靶基因的表达模式。软件综合预测可知miR-124-3p_4调控8个卵巢偏向性基因,其中与miR-124-3p_4互作能力最强的基因为dpgn-like基因;表达结果显示dpgn-like基因在卵巢中的表达显著高于精巢,且其表达定位于卵原和卵母细胞胞质;双荧光素酶报告基因分析以及体内过表达miRNA实验从体外和体内两个方面验证了miR-124-3p_4能够下调dpgn-like基因的表达。研究结果表明,miR-124-3p_4可以抑制卵巢高表达基因dpgn-like在精巢中的表达,暗示其可能在栉孔扇贝性腺发育中发挥调控作用。Scallops belong to bivalves of the mollusc phylum and are important shellfish culture species in China.Important varieties of scallops and their complex reproductive regulation mechanisms have been the focus of research in shellfish biology.Many genes have been reported to be involved in the sex differentiation and gonadal development of scallops,whereas the regulation of these genes has rarely been reported.MicroRNA is a class of important endogenous regulatory factors that can participate in the regulation of gene expression.To explore the role of microRNAs in the gonadal development of shellfish,this study took the Zhikong scallop(Chlamys farreri),a hermaphroditic and sex-stable bivalve species and thus good material for such a mechanism exploration,as the research material.In a previous study,we found that miR-124-3p_4 was highly expressed in the testes of juveniles during the early stage of sexual differentiation,and thus took this microRNA as the research object.The sequence of miR-124-3p_4 was compared with other species and the core sequence was consistent.The genes targeted by miR-124-3p_4 were predicted through RNAhybrid and miRanda,from which 729 and 436 target genes were obtained,respectively,and the intersection was 260.Combining with the transcriptome data of C.farreri,eight ovary bias genes and twelve testis genes were obtained.Based on the binding site and binding-free energy analysis,all eight ovary bias genes matched perfectly with miR-124-3p_4 and their free energy was all below–20 kcal/mol,indicating the interaction activities between them.Among these genes,dpgn-like was found to have the strongest interaction with miR-124-3p_4.Sequence and structure analyses of the dpgn-like gene showed that its cDNA full length was 4526 bp,coding 559 amino acids.Four MFS,two KAZAL_FS,and one OATP conserved domain were identified in the DPGNL protein.Phylogenetic results clarified its identity and evolutionary status.The expression of the dpgn-like gene was mainly in the mantle,foot,gill and gonads,of which

关 键 词：栉孔扇贝 miR-124-3p_4 dpgn-like基因 表达特征 

分 类 号：S917[农业科学—水产科学]