克氏原螯虾泛素结合酶E2r基因的克隆及表达分析

为了解泛素结合酶E2r基因在克氏原螯虾性腺发育中的作用,利用RACE技术得到了c DNA全序列,命名为Pc-UBE2r。该基因序列全长为3 671 bp,包含泛素结合酶的特有结构域,以及半胱氨酸残基活化位点。开放阅读框为729 bp,编码242个氨基酸,预测蛋白分子量约为27.6 ku。BLAST比对发现,克氏原螯虾UBE2r基因与节肢动物UBE2r基因具有较高的同源性。系统进化分析表明,Pc-UBE2r基因与日本囊对虾聚为一枝。qRT-PCR研究表明,Pc-UBE2r基因在性腺中的表达量显著高于其他组织(P0.05)。在卵巢中,PcUBE2r基因的表达量在未发育期最低,在卵黄发生前期表达量快速增长,随后表达量逐渐降低。在精巢中,Pc-UBE2r基因的表达量在精母细胞发生期达到最高水平。组织原位杂交结果显示,Pc-UBE2r基因在卵巢中均匀分布在未发育期的卵母细胞细胞质中,随后逐渐迁移到卵母细胞细胞核以及滤泡细胞周围。在精巢中,Pc-UBE2r基因主要分布在精母细胞的周围。组织总蛋白Western blot检测发现,UBE2r蛋白在精巢和卵巢中的表达量也显著高于其他组织(P0.05)。综上所述,我们推测Pc-UBE2r基因在克氏原螯虾配子发生和性腺发育方面发挥了重要作用,本研究将为虾蟹类性腺发育调控的分子机制奠定基础和提供依据。

Molecular cloning and expression analysis of ubiquitin-conjugating enzyme E2r in Procambarus clarkii

In our study, for a better understanding of the role regarding UBE2r in the development of gonad in crawfish, the full length cDNA of Procambarus clarkii UBE2r gene (Pc-UBE2r) were cloned and characterized. The full-length Pc-UBE2r cDNA sequence obtained was 3671 base pairs (bp) including 554 bp of 5''-UTR and 2 389 bp in 3''-UTR, while containing an open reading frame of 729 bp encoding a protein of 242 amino acids with a predicted molecular weight of 27.6 ku. Bioinformatics analysis indicated that the Pc-UBE2r gene had a conserved domain specified in ubiquitin proteasome family and Cys94 sites. The homology and phylogenetic analysis showed that the deduced amino acid sequence of UBE2r shared high homology in different species and the highest conservation with Marsupenaeus japonicus. Quantitative real-time PCR analysis revealed that Pc-UBE2r was distributed in various tissues, while its expression in the ovary and testis was significantly higher than other tissues (P<0.05). In the development of ovary, the expression of Pc-UBE2r was the lowest in the non-developmental stage, and increased rapidly in the previtellogenic stage. With the maturity of the ovary, the expression of Pc-UBE2r decreased gradually. Meanwhile, during the development of testis, the expression of Pc-UBE2r was proven highest in the spermatocyte generation stage. In our in situ hybridization experimental procedure, the positive signal of Pc-UBE2r was observed uniformly distributed in the oocyte cytoplasm in the non-developmental stage of the ovary. With the development of the ovary, the positive signals of Pc-UBE2r surrounded the oocyte nucleus and follicle cells can be found. In the testis, the positive signals were mainly distributed in the secondary spermatocytes. Besides, Western blotting analysis exhibited that the expression distributions of UBE2r in tissues were significantly different, but remained highl in ovary and testis (P<0.05). Overall, the experimental results demonstrated that Pc-UBE2r may play an important role in the gametogenesis and gonadal development of crawfish.