草鱼和赤眼鳟nanos2基因的克隆与表达特征分析
作者:
作者单位:

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

作者简介:

黄玲(1996-),女,硕士,研究实习员,研究方向为水生动物遗传育种与分子生物学.E-mail:huangling@yfi.ac.cn

中图分类号:

S917

基金项目:

农业生物育种重大专项(2023ZD4054);中国水产科学研究院中央级公益性科研院所基本科研业务费项目(2022YJ08,2022XT03, 2023TD23)


Cloning and expression characterization analysis of nanos2 gene in Ctenopharyngodon idella and Squaliobarbus curriculus
Author:
Affiliation:

Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture and Rural Affairs of China, YangtzeRiver Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223 , China

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    摘要:

    草鱼(Ctenopharyngodon idella)是我国重要的经济鱼类。为了加快草鱼育种进程, 笔者建立了草鱼生殖干细胞移植方法, 利用赤眼鳟(Squaliobarbus curriculus)借腹生殖产生草鱼配子。为建立移植后鉴定草鱼和赤眼鳟生殖细胞的方法, 本研究克隆得到了草鱼和赤眼鳟生殖相关基因 nanos2 的全长 cDNA 序列, 对其进行序列分析, 通过荧光定量 PCR (qPCR)研究其在不同组织的表达水平, 最终设计特异性引物区分草鱼和赤眼鳟生殖细胞。克隆的草鱼和赤眼鱒 nanos2 cDNA 序列长度分别为 649 bp 和 636 bp, 分别编码 145 和 144 个氨基酸; 比对发现草鱼和赤眼鳟 Nanos2 氨基酸序列同源性高达 91.67%, 与斑马鱼(Danio rerio)具有 65.49%同源性; 系统发育进化树显示草鱼和赤眼鳟的亲缘关系最近; 荧光定量 PCR 分析表明, nanos2 基因主要在草鱼和赤眼鳟性腺表达, 且精巢的表达水平显著高于卵巢; 通过比对草鱼和赤眼鳟 nanos2 基因序列, 设计获得草鱼和赤眼鳟特异性与通用引物, PCR 结果显示, 草鱼 nanos2 特异性引物只在草鱼性腺扩增出 179 bp 的目的条带, 赤眼鳟 nanos2 特异性引物只在赤眼鳟性腺扩增出 265 bp 的目的条带, nanos2 通用引物在草鱼和赤眼鳟性腺都能扩增出 251 bp 的目的条带, 结果表明利用特异性引物可以高效区分草鱼和赤眼鳟生殖细胞。本研究为进一步探讨 nanos2 在草鱼和赤眼鳟性腺发育的作用机制奠定基础, 也为后续监测草鱼生殖细胞在赤眼鳟性腺的嵌合及发育情况提供了一种有效方法。

    Abstract:

    The grass carp (Ctenopharyngodon idella) represents an economically important fish species in freshwater aquaculture within China. However, the urgency to develop superior varieties has become paramount due to significant germplasm degeneration in grass carp. Germline stem cell transplantation emerges as a promising and potent technique for reducing the sexual maturity cycle in fish. Consequently, our objective was to shorten the generation interval for obtain functional gametes of grass carp by employing barbel chub (Squaliobarbus curriculus) as a surrogate host. The accurate identification of germ cells between donor and recipient species is a critical step in the successful establishment of germline stem cell transplantation. However, the method of identification of germ cells in grass carp and barbel chub is not clear. In the present study, the full-length cDNA of reproduction-related gene nanos2 were cloned from grass carp and barbel chub, which are 649 and 636 base pairs, encoding 145 and 144 amino acids, respectively. Sequence analysis revealed that the grass carp Nanos 2 (CiNanos2) amino acid sequence exhibits a high degree of sequence identity to that of barbel chub (91.67%), and to that of zebrafish (Danio rerio) (65.49%). Phylogenetic tree analysis showed that grass carp is clustered together with barbel chub, indicating the closest genetic relationship between them. The expression of nanos2 transcripts in grass carp and barbel chub was predominantly observed in the gonads, with significantly higher levels detected in the testis compared to the ovary, suggesting that nanos2 might plays a crucial role in the development of gonad. Species-specific and common primers were designed based on the alignment of nanos2 sequences between grass carp and barbel chub for PCR analysis. The results demonstrated that the grass carp-specific primers had exclusively target product (179 bp) in the gonad of grass carp, while the barbel chub-specific primers had only expected product (265 bp) in the gonad of barbel chub gonads. Additionally, the common primers were able to amplify indiscriminately in the gonads of both grass carp and barbel chub, producing a target product of 251 bp, implying that the germ cells of grass carp and barbel chub could be efficiently distinguished via species-specific primers by PCR. Our study laid the foundation for further investigating the mechanism of the nanos2 gene in gonad development of grass carp and barbel chub. Meanwhile, it provided an effective method for monitoring the chimerism and development of grass carp germ cells in the gonads of barbel chub.

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黄玲,叶欢,岳华梅,侯彦岭,屈紫玲,阮瑞,李创举.草鱼和赤眼鳟nanos2基因的克隆与表达特征分析[J].中国水产科学,2024,31(12):1411-1421
HUANG Ling, YE Huan, YUE Huamei, HOU Yanling, QU Ziling, RUAN Rui, LI Chuangju. Cloning and expression characterization analysis of nanos2 gene in Ctenopharyngodon idella and Squaliobarbus curriculus[J]. Journal of Fishery Sciences of China,2024,31(12):1411-1421

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  • 收稿日期:2024-08-15
  • 最后修改日期:2024-09-18
  • 在线发布日期: 2025-02-11
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