克氏原螯虾泛素结合酶E2基因的克隆及其在卵巢中的表达分析

Cloning and expression analysis of ubiquitin-conjugating enzyme E2 gene of Procambarus clarkii

利用RACE技术获得了UB-E2基因c DNA全长,通过RT-PCR技术研究UB-E2基因在不同组织、发育时期的表达量和17α-羟孕酮激素刺激后UB-E2表达量及性腺发育情况。结果表明:该基因序列全长3 943bp,开放阅读框675 bp,编码224个氨基酸,相对分子量23. 93 ku,等电点8. 61,82～219位氨基酸序列为泛素结合酶家族特有的结构域。氨基酸序列比对发现,克氏原螯虾UB-E2基因与节肢动物UB-E2基因有较高的同源性;系统进化分析表明,UB-E2基因与凡纳滨对虾和斑节对虾聚为一支。荧光定量结果显示,UB-E2基因在克氏原螯虾卵巢组织中的表达量显著高于其他组织(P <0. 05);在卵巢发育初期表达量最低,随后逐渐升高,到产卵后期开始下降; 17α-羟孕酮刺激结果显示,试验组卵巢发育速度明显加快,同时UB-E2基因的表达量也显著上升,卵巢发育速度和UB-E2基因表现为受17α-羟孕酮刺激响应一致。推测UB-E2基因在克氏原螯虾的卵巢发育和配子发生中起着重要的作用,本研究为克氏原螯虾性腺发育分子调控机制提供一定的理论依据。

In this study,the full-length cDNA of UB-E2was obtained using RACE technology in Procambarus clarkii.The expression of UB-E2gene in different tissues and development stages and the expression of UB- E2and gonadal development after hormone stimulation by 17α-hydroxyprogesterone were studied by RT-PCR. The results showed that the gene has a full-length sequence of 3943bp,an open reading frame of 675bp, encoding 224amino acids,a relative molecular weight of 23.93ku,and an isoelectric point of 8.61.Its amino acid sequence from 82to 219is unique protein domain of the ubiquitin-conjugating enzyme family.The amino acid sequence alignment showed that UB-E2gene of Procambarus clarkii had higher homology with arthropod UB-E2gene.Phylogenetic analysis showed that the UB-E2gene was clustered with L.vannamei and P.monodon.Quantitative fluorescence results showed that the expression of UB-E2gene in ovary tissue of Procambarus clarkii was significantly higher than the other tissues (P <0.05);it was the lowest in the early stage of ovarian development,then it gradually increased,and it began to decline in the late spawning period;17α-hydroxyprogesterone stimulation results showed that the ovarian development of the experimental group was significantly accelerated,while the UB-E2gene expression was also significantly increased,ovarian development and UB-E2gene expression were consistent with 17α-hydroxyprogesterone stimulated response. It is speculated that UB-E2gene plays an important role in ovarian development and gametogenesis in Procambarus clarkii.This study will provide a theoretical basis for the molecular regulation of gonad development in Procambarus clarkii.