Zhao C, Fu M J, Zhou F L, Yang Q B, Jiang S G, Qiu L H
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China.
South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China
Genet Mol Res. 2015 Oct 27;14(4):13380-90. doi: 10.4238/2015.October.26.36.
The open reading frame of black tiger shrimp (Penaeus monodon) cyclin B (Pmcyclin B) was identified, based on cDNA sequence registered in GenBank (accession No. EF015590). The target sequence was 1206 bp, corresponding to 401 amino acids. Two conserved signature sequences of the cyclin B gene family were found in the Pmcyclin B deduced aa sequence. Temporal expression of Pmcyclin B in different tissues, including ovary, lymphoid organ, brain, blood, muscle, heart, gill, hepatopancreas, and intestine, were quantified by quantitative real time PCR. Messenger RNA expression levels of Pmcyclin B were greatest in the ovary, compared to other tissues (P < 0.05). Temporal expression of Pmcyclin B in the ovary at six different developmental stages was investigated by real-time PCR; no significant difference was observed (P < 0.05). Recombinant Pmcyclin B protein and its polyclonal antibody were successfully produced. Western blot analysis revealed differential expression of Pmcyclin B in ovaries in developmental stages II to IV; a positive signal (45 kDa) was observed in all ovarian stages assessed, but was most intense at stage III. Pmcyclin B protein was assessed by immunohistochemistry and was localized to the cytoplasm of prophase oocytes at stage II and enriched in the nuclei of pro-metaphase oocytes at stages III and IV. Results from this study indicate that Pmcyclin B is constitutively expressed and plays an important role in ovarian maturation in P. monodon.
基于GenBank中注册的cDNA序列（登录号EF015590），鉴定出了黑虎虾（斑节对虾）细胞周期蛋白B（Pmcyclin B）的开放阅读框。目标序列为1206 bp，对应401个氨基酸。在推导的Pmcyclin B氨基酸序列中发现了细胞周期蛋白B基因家族的两个保守特征序列。通过定量实时PCR对Pmcyclin B在不同组织（包括卵巢、淋巴器官、脑、血液、肌肉、心脏、鳃、肝胰腺和肠道）中的时序表达进行了定量分析。与其他组织相比，Pmcyclin B的信使RNA表达水平在卵巢中最高（P < 0.05）。通过实时PCR研究了Pmcyclin B在卵巢六个不同发育阶段的时序表达；未观察到显著差异（P < 0.05）。成功制备了重组Pmcyclin B蛋白及其多克隆抗体。蛋白质印迹分析显示，Pmcyclin B在发育阶段II至IV的卵巢中存在差异表达；在所有评估的卵巢阶段均观察到阳性信号（45 kDa），但在阶段III最为强烈。通过免疫组织化学对Pmcyclin B蛋白进行了评估，结果显示其在II期前期卵母细胞的细胞质中定位，在III期和IV期前中期卵母细胞的细胞核中富集。本研究结果表明，Pmcyclin B持续表达，并在斑节对虾卵巢成熟过程中发挥重要作用。
