Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, 201306, China.
Shanghai Engineering Research Center of Aquaculture, Shanghai Ocean University, Shanghai, 201306, China.
Mol Biol Rep. 2022 Jul;49(7):6601-6611. doi: 10.1007/s11033-022-07501-2. Epub 2022 May 26.
Cyclin D (CCND) plays an important role in the cell cycle and is a rate-limiting factor that facilitates the G1/S transition.
In this study, the full-length cDNA of Hc-CCND2 was isolated from freshwater pearl mussel (Hyriopsis cumingii; Hc) and amplified using the 3´/5´ RACE system. The Hc-CCND2 expression profiles were analysed by quantitative real-time PCR. Functional analysis of the Hc-CCND2 genes was examined by both RNA interference (RNAi) and overexpression in H. cumingii.
Hc-CCND2 protein sequences were 295 amino acids long, possessed D-type cyclin signature motifs and contained conserved cyclin box domains. Hc-CCND2 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonad), with the highest expression levels found in the gill (P < 0.05). During the different developmental periods of the embryo, the relative expression of Hc-CCND2 increased with embryonic development, peaking at the blastula stage and decreasing significantly in the gastrula stage. After knockdown of Hc-CCND2 by RNAi, a significant decrease in CDK6 expression levels was found, while the percentage of cells in the G0/G1 phase significantly increased. Overexpression of Hc-CCND2 in mantle cells led to increased proliferation of cultured cells (P < 0.05).
Our results demonstrated that Hc-CCND2 may promote cell cycle progression in H. cumingii, and that overexpression of Hc-CCND2 promotes mantle cell proliferation. These findings may provide a novel approach for improving the slow proliferation rate of shellfish cells in in vitro cultures.
细胞周期蛋白 D（CCND）在细胞周期中起着重要作用，是促进 G1/S 期过渡的限速因子。
本研究采用 3'/5' RACE 系统从淡水珍珠贝（Hyriopsis cumingii；Hc）中分离全长 cDNA 的 Hc-CCND2，并进行扩增。采用实时定量 PCR 分析 Hc-CCND2 的表达谱。通过 RNA 干扰（RNAi）和在 H. cumingii 中的过表达来检测 Hc-CCND2 基因的功能分析。
Hc-CCND2 蛋白序列长 295 个氨基酸，具有 D 型细胞周期蛋白特征基序，并含有保守的细胞周期蛋白盒结构域。Hc-CCND2 在所有检测的组织（贻贝、足、内脏团、鳃、外套膜、外套膜和性腺）中均有表达，其中在鳃中的表达水平最高（P<0.05）。在胚胎的不同发育时期，Hc-CCND2 的相对表达随着胚胎发育而增加，在囊胚期达到峰值，并在原肠胚期显著下降。通过 RNAi 敲低 Hc-CCND2 后，CDK6 表达水平显著降低，而 G0/G1 期的细胞比例显著增加。过表达 Hc-CCND2 在外套膜细胞中导致培养细胞增殖增加（P<0.05）。
我们的结果表明，Hc-CCND2 可能促进 H. cumingii 中的细胞周期进程，而过表达 Hc-CCND2 促进外套膜细胞增殖。这些发现可能为提高贝类细胞在体外培养中的增殖速度提供新方法。
