Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou 515063, China; STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantou 515063, China.
STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantou 515063, China; Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, Kuala Nerus 21030, Malaysia.
Int J Biol Macromol. 2021 Jul 31;183:490-501. doi: 10.1016/j.ijbiomac.2021.04.186. Epub 2021 May 3.
The wingless-type MMTV integration site family member-4 (Wnt4), a member of the wingless-related integration site (Wnt) family, is widely accepted as a key regulator of ovarian development in mammals. In this study, a full-length cDNA of Wnt4 (designated as Sp-Wnt4) was cloned, characterized, and functionally studied in mud crab (Scylla paramamosain). The full-length cDNA of Sp-Wnt4 consists of 2659 bp with an open reading frame (ORF) encoding 359 amino acids, a 907 bp 5'-UTR and a 672 bp 3'-UTR. Sp-Wnt4 contains 25 cysteine (Cys) residues and three potential N-glycosylation sites. Sp-Wnt4 protein shared the highest identity (98.9%) to the Wnt4 protein of Portunus trituberculatus. The phylogenetic tree showed that Sp-Wnt4 and Wnt4 protein of Malacostracan crustaceans clustered together, indicating that they had a close genetic distance. Sp-Wnt4 was expressed at a higher level in the ovary compared to other tissues, with the highest expression level at the third stage (O-III) of the ovarian development (P < 0.05). A downward trend was observed in the expression level of Sp-Wnt4 from the embryo stage to crablet stages (P < 0.05). After unilateral eyestalk ablation, the expression level of Sp-Wnt4 significantly increased in testis (14-fold) and downregulated (3.1-fold) in the gill (P < 0.05) of females. In situ hybridization (ISH) assay revealed that Sp-Wnt4 transcripts were mainly localized in the cytoplasm of oocyte cells. These findings showed that Sp-Wnt4 play crucial roles in the ovarian development of S. paramamosain. In conclusion, our study provides novel insights into the evolution and roles of the Wnt4 gene.
无翅型 MMTV 整合位点家族成员 4（Wnt4）是无翅相关整合位点（Wnt）家族的成员，被广泛认为是哺乳动物卵巢发育的关键调节因子。本研究克隆并鉴定了泥蟹（Scylla paramamosain）全长 Wnt4 cDNA（命名为 Sp-Wnt4），并对其功能进行了研究。Sp-Wnt4 的全长 cDNA 由 2659 bp 组成，包含一个编码 359 个氨基酸的开放阅读框（ORF）、一个 907 bp 的 5'-UTR 和一个 672 bp 的 3'-UTR。Sp-Wnt4 含有 25 个半胱氨酸（Cys）残基和三个潜在的 N-糖基化位点。Sp-Wnt4 蛋白与三疣梭子蟹（Portunus trituberculatus）的 Wnt4 蛋白具有最高的同源性（98.9%）。系统进化树显示，Sp-Wnt4 和十足目甲壳动物的 Wnt4 蛋白聚为一簇，表明它们具有较近的遗传距离。Sp-Wnt4 在卵巢中的表达水平明显高于其他组织，在卵巢发育的第三阶段（O-III）表达水平最高（P<0.05）。从胚胎期到幼蟹期，Sp-Wnt4 的表达水平呈下降趋势（P<0.05）。单侧眼柄切除后，Sp-Wnt4 在雌蟹的精巢中表达水平显著升高（14 倍），在鳃中表达水平下调（3.1 倍）（P<0.05）。原位杂交（ISH）检测显示，Sp-Wnt4 转录本主要定位于卵母细胞的细胞质中。这些结果表明，Sp-Wnt4 在泥蟹卵巢发育中发挥着关键作用。综上所述，本研究为 Wnt4 基因的进化和功能提供了新的见解。
