| 本文已被：浏览 1597次 下载 125次 |
码上扫一扫！ |
| 大鳍鳠 dnd 基因的克隆与表达特征分析 |
|
余乐1, 王锐1, 叶欢2, 岳华梅2, 谢勇3, 易沫4, 陈元坤3, 李创举1,2
|
|
1.武汉轻工大学 动物科学与营养工程学院，湖北 武汉 430023;2.中国水产科学研究院 长江水产研究所 农业农村部淡水生物多样性保护重点实验室，湖北 武汉 430223;3.重庆市水产科学研究所，重庆 400020;4.石首市农业技术推广中心，湖北 石首 434400
|
|
| 摘要: |
| 【 目的】探究鱼类生殖细胞特异标记基因dnd在大鳍鳠性腺发育过程中的表达特征。【 方法】采集 1龄、2龄和4龄雌雄大鳍鳠的心脏、肝脏、脾脏、肾脏、肠、脑、鳃及性腺组织卵巢和精巢，提取4龄大鳍鳠的卵巢总 RNA，
反转录合成 cDNA，采用RACE技术克隆大鳍鳠dnd基因（Hmdnd）全长cDNA序列，测序后进行分析；以 β-actin为内参基因，采用实时荧光定量 PCR（RT-qPCR）方法，检测dnd基因在1龄大鳍鳠不同组织以及不同发育时期性腺中的表达量；利用原位杂交技术分析dnd mRNA 在生殖细胞中的表达定位。【 结果】Hmdnd 基因cDNA序列全长2 035 bp（GenBank登录号为PQ626051），其中开放读码框1 131 bp，编码376个氨基酸。氨基酸序列比对和系统进化树分析结果表明，大鳍鳠与黄颡鱼dnd蛋白序列一致性最高（79.24%）。RT-qPCR结果表明，Hmdnd 基因在大鳍鳠性腺组织特异表达，且在卵巢的表达量显著高于精巢，在其他组织中不表达。随着配子发育的进行，Hmdnd基因表达量显著下降。性腺切片RNA原位杂交研究结果显示，Hmdnd基因主要在生殖细胞的细胞质表达；在卵巢中，随着初级卵母细胞从Ⅰ期发育到Ⅱ期，Hmdnd表达量急剧增加，但随着初级卵母细胞发育到Ⅲ期，Hmdnd表达量下降至Ⅰ期初级卵母细胞水平；在精巢中，Hmdnd在精原细胞中表达水平最高，随着精子发育的推进，其表达水平逐渐降低，在精子细胞中不表达。【 结论】Hmdnd基因可能在大鳍鳠性腺发育早期发挥着重要的作用。 |
| 关键词: 大鳍鳠 dnd基因 生殖细胞 性腺发育 |
| DOI：10.13207/j.jnwafu.2026.04.003 |
| 分类号: |
| 基金项目:重庆市科研机构绩效激励引导专项（CSTB2023JXJL-YFX0084）；重庆市财政项目（C20240103）；国家重点研发计划项目（2022YFD2400101）；中央级公益性科研院所基本科研业务费专项资金项目（2023TD23） |
|
| Cloning and expression analysis of the dnd gene in Hemibagrus macropterus |
|
YU Le1, WANG Rui1, YE Huan2, YUE Huamei2, XIE Yong3, YI Mo4, CHEN Yuankun3, LI Chuangju1,2
|
|
1.School of Animal Science and Nutritional Engineering，Wuhan Polytechnic University，Wuhan，Hubei 430023，China;2.Key Laboratory of Freshwater Biodiversity Conservation，Ministry of Agriculture and Rural Affairs，Yangtze River Fisheries Research Institute of Chinese Academy of Fishery Sciences，Wuhan，Hubei 430223，China;3.Chongqing Fisheries Science Research Institute， Chongqing 400020，China;4.Shishou Agricultural Technology Extention Center， Shishou，Hubei 434400，China
|
| Abstract: |
| 【 Objective】This study aimed to investigate the expression patterns of the germ cell-specific marker gene dead end （dnd） during gonadal development in Hemibagrus macropterus.【Method】Tissues in‐cluding the heart，liver，spleen，kidney，intestine，brain，gills，and gonads（ovary and testis） were collected from 1-，2-，and 4-year-old male and female H. macropterus. Total RNA was extracted from the ovaries of 4-yearold
H.macropterus，which was reverse-transcribed into cDNA，and the full-length cDNA sequence of H. macropterus dnd（Hmdnd） was cloned using RACE technology. Subsequently，sequence analysis was conducted. Using β-actin as the reference gene，real-time quantitative PCR（RT-qPCR） was employed to quantify dnd expression in various tissues of 1-year-old H. macropterus and in gonads at different developmental stages. RNA
in situ hybridization（ISH） was applied to localize dnd mRNA within germ cells.【Result】The full-length Hmdnd cDNA sequence was 2 035 bp（GenBank accession：PQ626051），containing an open reading frame （ORF） of 1 131 bp that encodes 376 amino acids. Sequence alignment and phylogenetic analysis revealed the highest protein sequence identity（79. 24%） between H. macropterus and Pelteobagrus fulvidraco（yellow cat‐fish）. RT-qPCR demonstrated that Hmdnd was exclusively expressed in gonads，with significantly higher levels in ovaries than in testes，and no expression in somatic tissues.Hmdnd expression declined markedly during gam‐ete maturation.RNA in ISH of gonadal sections revealed that Hmdnd mRNA was predominantly localized in the cytoplasm of germ cells. In ovaries，Hmdnd expression increased sharply as primary oocytes developed from stage Ⅰ to stage Ⅱ，but dropped to stage-Ⅰ-equivalent levels in stage Ⅲ primary oocytes. In testes，the stron‐gest Hmdnd signals were detected in spermatogonia and diminished progressively during spermatogenesis，with no expression observed in spermatozoa.【Conclusion】Hmdnd is likely to play a critical role in the early gonadal development of H. macropterus. |
| Key words: Hemibagrus macropterus dnd gene germ cell gonadal development |
