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(Larimichthyscrocea)性腺线性化cDNA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cDNA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441bp,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 uhiquitination is a fundamental post-translational modification event that serves as a signaling function in diverse biological processes,including cell-cycle progression,cell apoptosis,gene transcriptional regulation and expression,etc.In these processes of ubiquitin-proteasome pathway,ubiquitin-conjugating enzyme E2 is of pivotal importance that mediates more than 80 percent of protein degradation of eukaryotic cells,and it plays a key role on the specific selection of degraded of target protein.In this study,a homologous gene fragment of ubiquitin-conjugating enzymes E2 D4 (Lc-UBE2D4) was screened from previous constructed normalized gonad cDNA library of large yellow croaker Larimichthys crocea was obtained,and its full length cDNA was cloned by using SMART-RACE technology,and then the differential expression profiles in different tissues and different gonadal development stages were analyzed using quantitative real time PCR (qRT-PCR).The full length cDNA of Lc-UBE2D4 gene was of 798 bp,with 441 bp of ORF encoding a protein of 147 amino acids,and it contained the highly conserved ubiquitin-conjugating enzymes UBC domain and an active site cysteine.The qRT-PCR results revealed that the highest expression level of Lc-UBE2D4 presented in spleen and testis which were extremely higher than those in other tissues (P ＜ 0.01),and its expression level in ovary was significantly higher than other organs (excluding spleen and testis) (P ＜ 0.05).Moreover,its expression levels in three stages of testis were higher than in the same growth stages of ovary (P ＜ 0.05).These results suggested that Lc-UBE2D4 played essential roles in the gonad development,especially in testis development.Meanwhile,Lc-UBE2D4 may be involved in immune function due to highest expression in spleen.
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