凡纳滨对虾Lv-CDK17-like基因克隆与耐豆粕家系组织表达的研究

    Cloning of the Lv-CDK17-like gene in Litopenaeus vannamei and its tissue expression profile in soybean meal-tolerant families

    • 摘要:
      目的 克隆凡纳滨对虾Lv-CDK17-like基因,开展生物信息学分析,研究其在不同豆粕替代水平、遗传背景家系、发育阶段和组织类型中的表达特征,探究该基因在对虾应对植物蛋白饲料应激过程中的调控作用。
      方法 根据NCBI公布的序列设计特异性引物克隆Lv-CDK17-like基因编码区序列(CDS),对其氨基酸序列、蛋白结构域组成及系统发育关系进行分析;通过实时荧光定量PCR技术(qRT-PCR),检测该基因在20%(T20)和50%(T50)豆粕含量饲料饲喂条件下,3号和11号家系对虾不同发育时期中的表达差异。分别在0、15、30、45和60 d采集对虾肝胰腺、肠道和肌肉;从30 d起增加采集胃组织;并在60 d额外采集心、鳃和腹神经索,共计7个组织。
      结果 Lv-CDK17-like开放阅读框(ORF)长1 476 bp,编码491个氨基酸,预测蛋白分子量为55 kDa,理论等电点为8.98;生物信息学分析显示该蛋白含1个S_TKc结构域(162~443 aa),无跨膜结构域和信号肽;系统进化分析表明,凡纳滨对虾Lv-CDK17-like与日本沼虾亲缘关系最近,同源性达95.33%。表达分析揭示,在高豆粕替代(T50)条件下,Lv-CDK17-like基因在心脏和鳃组织表达量显著上调;不同家系呈现特异性表达模式,3号家系在T50条件下生长性能显著优于T20条件(T20最终体质量为3.2 ± 0.6 g,T50最终体质量为4.1 ± 0.4 g,体重增幅28.1%),且60 d时Lv-CDK17-like在心脏组织高表达;而11号家系在T20条件下表现最佳,T50处理下Lv-CDK17-like在胃组织表达显著抑制。
      结论 Lv-CDK17-like基因表达模式与家系生长性能、饲料适应性密切相关,可能在对虾应对植物蛋白饲料应激中发挥关键调控作用。本研究首次系统阐明Lv-CDK17-like在耐豆粕家系对虾中的组织表达特征,为水产养殖中高植物蛋白饲料的科学应用提供理论依据。

       

      Abstract:
      Objective This study aimed to clone the Lv-CDK17-like gene from Litopenaeus vannamei, perform bioinformatic analyses, and investigate its expression profiles across varying levels of soybean meal (SBM) replacement, genetic families, developmental stages, and tissue types, thereby exploring its regulatory role in the shrimp's response to plant-protein-induced dietary stress.
      Method Specific primers were designed based on the sequence deposited in NCBI to clone the coding sequence (CDS) of Lv-CDK17-like. Bioinformatic analyses were conducted to characterize its amino acid sequence, protein domain composition, and phylogenetic relationships. Real-time quantitative PCR (qRT-PCR) was employed to evaluate the expression differences of this gene in two shrimp families (Family No. 3 and Family No. 11) under two dietary treatments—20% (T20) and 50% (T50) SBM replacement—at multiple developmental time points. Samples were collected at days 0, 15, 30, 45, and 60; stomach tissue was additionally sampled from day 30 onward; and on day 60, heart, gill, and ventral nerve cord were also collected, totaling seven tissues.
      Result The open reading frame (ORF) of Lv-CDK17-like is 1 476 bp in length, encoding a 491-amino acid protein with a predicted molecular weight of 55 kDa and a theoretical isoelectric point (pI) of 8.98. Bioinformatic analysis reveal that the protein contains a single S_TKc domain (spanning residues 162-443), with no transmembrane domains or signal peptides. Phylogenetic analysis indicate that Lv-CDK17-like from L. vannamei is most closely related to that of Macrobrachium nipponense, with a sequence identity of 95.33%. Expression profiling demonstrate that Lv-CDK17-like is regulated by multiple factors: under high SBM replacement (T50), its expression is significantly upregulated in heart and gill tissues. Distinct expression patterns are observed between families—Family 3 exhibits significantly better growth performance under T50 than T20 (final body weight: 3.2 ± 0.6 g in T20 vs. 4.1 ± 0.4 g in T50, representing a 28.1% increase), accompanied by notably high expression in heart tissue at day 60; in contrast, Family 11 performs best under T20, and its expression is significantly suppressed in stomach tissue under T50.
      Conclusion The expression pattern of Lv-CDK17-like is closely associated with family-specific growth performance and dietary adaptability, suggesting a critical regulatory role in the shrimp's response to plant-protein dietary stress. This study provides the first systematic characterization of Lv-CDK17-like tissue expression profiles in SBM-tolerant shrimp families, offering theoretical support for the rational application of high plant-protein diets in aquaculture.