大黄鱼Lc-UBE2D4基因的克隆及其表达分析  

Cloning and Expression Profiles of Lc-UBE2D4 Gene from Large Yellow Croaker Larimichthys crocea

作　　者：韩坤煌 周鹏 邹志华 张子平 王艺磊 

机构地区：[1]集美大学水产学院农业部东海海水健康养殖重点实验室,厦门361021 [2]宁德市富发水产有限公司大黄鱼育种国家重点实验室,宁德352103

出　　处：《生物技术通报》2017年第11期166-173,共8页Biotechnology Bulletin

基　　金：福建省自然科学基金项目(2015J06019);福建省STS项目(2016T3041);福建省海洋生物资源开发利用协同创新中心产学研基金(FJMBIO1502);大黄鱼育种国家重点实验室开放课题基金(LYC2016RS02);厦门南方海洋研究中心资助项目(14GZP75NF39)

摘　　要：泛素缀合酶E2是蛋白质泛素化系统中的关键酶,对降解的靶蛋白的特异性选择起决定性作用,参与了细胞内80%以上的蛋白降解,对于细胞周期调控、细胞凋亡、基因转录及表达等生理活动具有重要的调控作用。本研究从已构建的大黄鱼(Larimichthys crocea)性腺线性化c DNA文库中筛选出泛素缀合酶(Ubiquitin-conjugating enzymes E2 D4,Lc-UBE2D4)同源基因片段,利用SMART-RACE方法克隆出其全长c DNA序列,并利用荧光定量PCR技术检测其在大黄鱼不同组织和性腺不同发育阶段的差异表达情况。结果显示,Lc-UBE2D4基因全长798 bp,其中ORF为441 bp,可编码147个氨基酸,含有一段典型的泛素缀合酶UBC结构域及其半胱氨酸激活位点。定量PCR结果分析发现,Lc-UBE2D4在脾脏和性腺中表达量较高,在脾脏和精巢的表达水平极显著高于其他各组织(P<0.01),同时在卵巢中的表达亦显著高于除脾脏和精巢外的其他各组织(P<0.05);通过对Lc-UBE2D4基因在性腺不同发育阶段表达模式的研究发现,该基因在精巢3个时期中的表达水平显著高于各发育阶段的卵巢(P<0.05),推测Lc-UBE2D4基因在大黄鱼性腺发育过程中起着重要的调节作用,脾脏中的高表达也暗示其可能参与免疫机能。Protein ubiquitination is a fundamental post-translational modification event that serves as a signaling function in diversebiological processes,including cell-cycle progression,cell apoptosis,gene transcriptional regulation and expression,etc. In these processesof ubiquitin-proteasome pathway,ubiquitin-conjugating enzyme E2 is of pivotal importance that mediates more than 80 percent of proteindegradation of eukaryotic cells,and it plays a key role on the specific selection of degraded of target protein. In this study,a homologous genefragment of ubiquitin-conjugating enzymes E2D4（Lc-UBE2D4）was screened from previous constructed normalized gonad cDNA library oflarge yellow croaker Larimichthys crocea was obtained,and its full length cDNA was cloned by using SMART-RACE technology,and thenthe differential expression profiles in different tissues and different gonadal development stages were analyzed using quantitative real time PCR（q RT-PCR）. The full length cDNA of Lc-UBE2 D4 gene was of 798 bp,with 441 bp of ORF encoding a protein of 147 amino acids,and itcontained the highly conserved ubiquitin-conjugating enzymes UBC domain and an active site cysteine. The q RT-PCR results revealed that thehighest expression level of Lc-UBE2 D4 presented in spleen and testis which were extremely higher than those in other tissues（P〈 0.01）,andits expression level in ovary was significantly higher than other organs（excluding spleen and testis）（P〈 0.05）. Moreover,its expressionlevels in three stages of testis were higher than in the same growth stages of ovary（P 〈0.05）. These results suggested that Lc-UBE2 D4 playedessential roles in the gonad development,especially in testis development. Meanwhile,Lc-UBE2 D4 may be involved in immune function dueto highest expression in spleen.

关 键 词：泛素化 Lc-UBE2D4 大黄鱼 组织表达 性腺发育 

分 类 号：Q78[生物学—分子生物学] S917.4[农业科学—水产科学]