许氏平鲉蛋白激酶B (SsAkt)基因的克隆及其mRNA在细菌胁迫后的表达规律

Cloning of SsAkt in Sebastes schlegelii and its expression pattern after bacterial stimulation

中文关键词: 许氏平鲉  蛋白激酶B  Akt  基因克隆  表达规律

英文关键词: Sebastes schlegelii  Protein kinase B  Akt  Gene cloning  Expression pattern

中文摘要:

      为研究蛋白激酶B (Akt)在许氏平鲉(Sebastes schlegelii)应答细菌胁迫过程中的作用，本研究应用PCR技术对许氏平鲉Akt基因(SsAkt)的编码区序列进行了克隆和特征分析，并应用实时荧光定量PCR技术检测了健康许氏平鲉各组织和细菌胁迫后肾脏、血液和肝脏中SsAkt mRNA的表达规律。结果显示，SsAkt的开放阅读框(ORF)的长度为1 440 bp，编码479个氨基酸，预测其蛋白的相对分子质量为55.80 kDa，理论等电点(pI)为5.64。SMART分析显示，SsAkt蛋白含有丝氨酸/苏氨酸蛋白激酶家族的3个特征性保守结构域和2个磷酸化位点。同源比对发现，SsAkt与鱼类的同源性较高，与尖嘴鲈(Lates calcarifer)的相似度最高，为99.37%。组织表达分析显示，SsAkt在许氏平鲉健康组织(血液、鳃、肝脏、肌肉、肾脏、脾脏、肠、脑和心脏)中均有表达，在肾脏、脑和血液中的相对表达量较高。许氏平鲉响应细菌胁迫的表达结果显示，藤黄微球菌(Micrococcus luteus)感染后，SsAkt在3种组织中的表达明显上调，呈先上升后下降的趋势；鳗弧菌(Vibrio anguillarum)感染后，SsAkt在3种组织中呈现不同的表达趋势：在肾脏中，SsAkt的表达趋势为先上升后下降又上升再下降，而在血液和肝脏中的表达趋势为先上升后下降。以上研究表明，SsAkt响应了外源微生物对许氏平鲉的胁迫，在抵御外源微生物免疫应答过程中发挥重要作用。本研究结果为许氏平鲉的免疫机理研究奠定了基础。

英文摘要:

      Sebastes schlegelii is a cold temperate ovoviviparous fish found near the seabed layer and is widely distributed in the Yellow Sea and Bohai Sea of China, as well as in the coasts of South Korea and Japan. It is characterized by its tender meat, delicious taste, rich nutrition, and high medicinal value, thus it is an economically important marine fish species in the northern coastal areas of China. In recent years, deep-water cage cultures have rapidly developed in China. Among the economically important fish species suitable for cage culture in northern China, S. schlegelii is the most important local breed with excellent quality and characteristics. However, with the expansion of the scale of the aquaculture industry, aquaculture diseases including those caused by Vibrio harveyi, Vibrio anguillarum, and Photobacterium damselae subsp. damselae, occur frequently. The authors isolated and identified a dominant pathogenic bacterium, the P5W of V. harveyi, from the lesions of diseased S. schlegelii and studied the response of S. schlegelii to V. harveyi infection. Multitissue transcriptome analysis screened the immune differential gene protein kinase B. Protein kinase B, also known as Akt protein, is a serine or threonine protein kinase, which mainly conducts signal transduction by phosphorylating other proteins. It plays an important role in various biological processes such as cell metabolism, transcriptional regulation, cell cycle regulation, immune defense, and embryonic development. Many studies have shown that during viral infection of cells, the Akt pathway is activated, thereby inhibiting viral proliferation. To clarify the sequence information, evolutionary characteristics, and biological functions of the Akt gene in S. schlegelii and to understand the immune response mechanism of S. schlegelii to bacterial invasion, the full-length cDNA of the SsAkt gene was obtained using molecular biology, and the sequence characteristics, tissue expression rules of the gene, and the response rules to Micrococcus luteus and V. anguillarum were studied. The results showed that the length of the open reading frame was 1 440 bp. The predicted relative molecular mass of the SsAkt protein was 55.80 kDa and the isoelectric point was 5.64. Sequence analysis revealed that the SsAkt protein contains three conserved domains: the PH domain (residues 6–109), serine/threonine protein kinase catalytic (S_TKc) domain (residues 148–405), and serine/threonine protein kinase (S_TK_X) domain (residues 406–475). The conserved amino acid site appears in the center of the S_TKc domain and at the end of the S_TK_ X domain: Thr305 and Ser472. Homologous alignment showed that the amino acid sequence of this gene was highly conserved among different species. Phylogenetic tree analysis showed that SsAkt is closely related to Akt in vertebrates, such as Sebastes umbrosus, Lates calcarifer, Larimichthys crocea, and Cyprinus carpio. The relative expression of SsAkt in different tissues of S. schlegelii was detected by real-time fluorescence quantitative PCR. The results showed that SsAkt was expressed in all tested healthy tissues of S. schlegelii, which indicated the relative expression of the SsAkt gene in S. schlegelii. This expression has distinct tissue specificity. The expression of SsAkt in the kidney is the highest, and in the blood and brain, it is also higher than that in other tissues. Therefore, it is speculated that higher expression of SsAkt in the kidney and brain is closely related to immune defense, neurodevelopment, and other processes. To study the role of SsAkt in the immune response of S. schlegelii, we designed a bacterial challenge experiment. The results showed that the transcription level of SsAkt changed significantly, and the relative expression of SsAkt in the three tissues increased after stimulation with M. luteus and V. anguillarum compared with the control group (PBS). After infection with M. luteus, its expression only increased sharply in the kidney, whereas its expression increased slowly in the blood and liver. In the experimental group infected with V. anguillarum, the expression increased significantly in the liver and kidney, and the two relative expression peaks in the kidney occurred at 12 h and 72 h after infection, respectively. The results revealed that the SsAkt gene may play an important role in the immune defense of S. schlegelii, which can further enrich the sequence information and evolutionary data of the Akt gene in marine fish and provide reference materials for an in-depth study of the biological function of this gene. In follow-up experiments, we will explore the specific mechanism of SsAkt in the innate immune defense of S. schlegelii to provide theoretical guidance for the study of the immune signaling pathway of S. schlegelii and to lay a theoretical foundation for revealing the role of SsAkt in other important biological functions in growth and development.