凡纳滨对虾6-磷酸海藻糖合成酶在抗高温胁迫中的表达特征

Expression Characteristics of Trehalose-6-Phosphate Synthase in Litopenaeus vannamei under High-Temperature Stress

中文关键词: 6-磷酸海藻糖合成酶  凡纳滨对虾  高温胁迫  基因表达

英文关键词: Trehalose-6-phosphate synthase  Litopenaeus vannamei  High temperature stress  Gene expression

中文摘要:

      6-磷酸海藻糖合成酶(Trehalose-6-phosphate synthase, TPS)是海藻糖合成的关键酶，在生物体逆境胁迫应答中发挥着重要的作用。本研究以凡纳滨对虾(Litopenaeus vannamei)高温胁迫转录组测序的Unigene序列为基础，采用直接PCR扩增的方法，获得了TPS部分cDNA (完整的ORF和部分UTR)序列(LvTPS)。序列分析结果显示，LvTPS序列包含1个2529 bp的开放阅读框，可编码842个氨基酸，分子量为95.4 kDa，等电点为6.17。LvTPS具有Glyco-transf-20和Trehalose PPase 2个功能结构域。多序列比对结果显示，LvTPS与中国对虾(Fenneropenaeus chinensis)的相似性最高，为63.73%；系统进化树显示，凡纳滨对虾与中国对虾亲缘关系最近，并与蓝蟹(Callinectes sapidus)、脊尾白虾(Exopalaemon carinicauda)、克氏原螯虾(Procambarus clarkia)等无脊椎动物聚为一支，脊椎动物单独聚为一支。基因表达水平的定量分析结果显示，LvTPS在鳃、肝胰腺、眼柄、心脏、神经和肌肉6种组织中均表达。鳃和肌肉表达量基本相同，为最高；眼柄、心脏和神经表达量次之，显著低于鳃和肌肉中的表达量(P<0.05)；肝胰腺中表达量为最低，显著低于其他5种组织中的表达量(P<0.05)。与26℃温度下的凡纳滨对虾相比，水温升至32℃时，眼柄和心脏中的LvTPS显著上调表达(P<0.05)；水温升至38℃时，鳃、肝胰腺、眼柄和心脏中LvTPS显著上调表达(P<0.05)。其中，在肝胰腺中表达量变化较显著。之后，随着温度的下降，其表达量总体呈下调趋势。回温至32℃和26℃时，6种组织中LvTPS基因表达量与对照组均无显著性差异。在神经和肌肉中，不同温度胁迫下的LvTPS基因表达量没有显著变化。上述研究结果表明，LvTPS与凡纳滨对虾应对高温胁迫过程密切相关。本研究可为解析凡纳滨对虾应答高温胁迫机制提供一些基础数据。

英文摘要:

      Trehalose-6-phosphate synthase (TPS) is a key enzyme in trehalose synthesis and plays an important role in the response of organisms to stress. In this study, based on the unigene sequences from transcriptome sequencing of Litopenaeus vannamei challenged with high-temperature stress, the partial TPS cDNA sequence (complete ORF and partial UTR) (LvTPS) was obtained directly by PCR amplification. Sequence analysis revealed that the LvTPS sequence contained a 2529 bp open reading frame, encoding 842 amino acids with a molecular weight of 95.4 kDa and an isoelectric point of 6.17. LvTPS had two functional domains, the Glyco-transf-20 domain and the Trehalose_PPase domain. Multiple sequence alignments showed that LvTPS had the highest homology with the corresponding gene in Fenneropenaeus chinensis, with a similarity value of 63.73%. The phylogenetic tree indicated that LvTPS was closely related to that of F. chinensis, and clustered with invertebrates, such as Callinectes sapidus, Exopalaemon carinicauda, and Procambarus clarkia. Vertebrates were clustered onto a signal branch. Quantitative analysis of gene expression levels showed that LvTPS was widely expressed in all of the examined tissues with different expression levels. The gill and muscle had similar levels and the highest expression. The expression of the eye stalk, heart, and nerve was next, being significantly lower than that of gill and muscle (P<0.05). The expression in the hepatopancreas was the lowest, being significantly lower than that of the other five tissues (P<0.05). L. vannamei kept at 26℃ were used as control group. When the temperature was increased to 32℃, the LvTPS gene in the eye stalk and heart was significantly increased (P<0.05). When the temperature was increased to 38℃, the expression of the LvTPS gene significantly rose in gill, hepatopancreas, eye stalk, and heart (P<0.05). Expression in the hepatopancreas was significantly changed. Thereafter, the expression of the LvTPS gene decreased when the temperature decreased. When the temperature was returned to 32℃ and 26℃, the expression of the LvTPS gene in the six tissues was not significantly different from that of the control group. There was no significant difference in expression under different levels of temperature stress in the nerve and muscle. The above results indicated that LvTPS was closely related to the response to high-temperature stress. This study provided basic data for the analysis of the mechanism of the response to high-temperature stress in L. vannamei.