摘要
泛素-蛋白酶体系统作为重要的蛋白质修饰途径,参与多个生物学过程,其中泛素结合酶E2作为关键酶可以决定泛素化对靶蛋白的影响。为了探究泛素结合酶E2在半滑舌鳎雌性性腺发育中的作用,本研究克隆了半滑舌鳎Cs-UBE2D4基因的开放阅读框ORF,检测了该基因在不同组织和不同发育阶段性腺中的表达模式;构建了其启动子报告基因载体(pGL3-Cs-UBE2D4)并进行了启动子活性分析。Cs-UBE2D4开放阅读框为360 bp,编码119个氨基酸,含有保守的泛素结合酶E2结构域。荧光定量PCR结果显示,Cs-UBE2D4在雌鱼的各组织中广泛表达,其中在卵巢和肝脏中表达量较高,而在雄鱼中几乎检测不到表达;在卵巢各发育阶段中,该基因的表达量从90日龄开始上调,6月龄达到峰值,之后开始下调,在1.5龄时表达量最低,在3龄时表达量有所回升,其中6月龄表达量为90日龄表达量的1.97倍。双荧光素酶活性检测结果显示,Cs-UBE2D4启动子具有较强的转录活性。本实验将为进一步开展Cs-UBE2D4基因对半滑舌鳎卵巢发育和性别调控相关机制的研究提供理论依据。
The ubiquitin-proteasome system is an important protein modification pathway,involved in many biological processes.Ubiquitin-conjugating enzyme E2 is the key enzyme in regulating protein ubiquitination.In order to explore the function of ubiquitin-conjugating enzyme E2 in female gonad development of Cynoglossus semilaevis,the Open Reading Frame(ORF)of Cs-UBE2D4 gene was cloned in this study.Its expression pattern was measured in various tissues of male and female adults,and at different developmental stages of gonads as well.Then,we constructed Cs-UBE2D4 promoter reporter vector(pGL3-Cs-UBE2D4)and detected its activity.The ORF of Cs-UBE2D4 gene was 360 bp,encoding 119 amino acids with a conserved ubiquitin-conjugating enzyme E2 domain.qPCR results showed that Cs-UBE2D4 was widely expressed in all tissues of females,especially in ovary and liver,but it was hardly detected in males.The gene expression increased from 90 days post-hatcing(dph),peaked at 6 months post-hatcing(mph),and decreased afterwards.Its expression level was lowest at 1.5 years post-hatching(yph)and increased at 3 yph.During this period,its expression was 1.97-fold higher at 6 mph than that at 90 dph.Dual luciferase assay showed strong activity of the promoter of Cs-UBE2D4 gene,indicating its participation in transcription.This study provides a theoretical basis for future research on Cs-UBE2D4 gene in ovarian development and gender-related mechanisms of Cynoglossus semilaevis.
作者
程鹏
陈张帆
徐文腾
王娜
杨倩
巩志宏
崔忠凯
胡元日
陈松林
CHENG Peng;CHEN Zhangfan;XU Wenteng;WANG Na;YANG Qian;GONG Zhihong;CUI Zhongkai;HU Yuanri;CHEN Songlin(School of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266071,China;Shandong Key Laboratory of Marine Fisheries Biotechnology and Genetic Breeding,Qingdao 266071,China;College of Marine Life Sciences,Ocean University of China,Qingdao 266071,China)
出处
《水产学报》
CAS
CSCD
北大核心
2023年第3期27-36,共10页
Journal of Fisheries of China
基金
国家自然科学基金(31730099)
中央级公益性科研院所基本科研业务费专项(2020TD20)
泰山学者攀登计划项目。
