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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(P<0.05)。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(P<0.05)。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。
In order to understand the function of ubiquitin-binding enzyme E2r gene in gonadal development of Procambarus clarkii, a full-length cDNA sequence of PcDNA was obtained by RACE technique and named Pc-UBE2r. The full length of this gene is 3 671 bp, which contains the unique domain of ubiquitin-binding enzyme and the cysteine residue activation site. The open reading frame was 729 bp encoding a polypeptide of 242 amino acids with a predicted molecular weight of 27.6 ku. BLAST comparison showed that the UBE2r gene of Procambarus clarkii has higher homology with the arthropod UBE2r gene. Phylogenetic analysis showed that Pc-UBE2r gene was clustered with Japanese shrimp. qRT-PCR showed that the expression of Pc-UBE2r gene in gonads was significantly higher than that in other tissues (P <0.05). In the ovary, PcUBE2r gene expression in the undeveloped period of the lowest, in the early stage of the yolk expression rapid increase, then the expression decreased. In the testis, Pc-UBE2r gene expression reached the highest level in the spermatocyte. Tissue in situ hybridization results showed that Pc-UBE2r gene in the ovary evenly distributed in the oocyte cytoplasm in the developmental period, and then gradually migrated to the oocyte nuclei and follicular cells around. In the testis, Pc-UBE2r gene is mainly distributed around the spermatocytes. Tissue protein Western blot detection, UBE2r protein expression in the testis and ovary were significantly higher than other tissues (P <0.05). In summary, we speculate that Pc-UBE2r gene plays an important role in the development of gonads and gonadal development of C. przewalskii. This study will lay the foundation and provide the basis for the molecular mechanism of gonadal development and regulation in shrimp and crab.