| 吉富罗非鱼组蛋白酶B基因克隆及无乳链球菌感染后的表达分析 |
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| 引用本文： | 朱佳杰,敖秋桅,谭芸,罗永巨,蒋和生.吉富罗非鱼组蛋白酶B基因克隆及无乳链球菌感染后的表达分析[J].南方农业学报,2022,53(8):2294-2301. |
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| 作者姓名： | 朱佳杰 敖秋桅 谭芸 罗永巨 蒋和生 |
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| 作者单位： | 1. 广西水产科学研究院, 广西南宁 530021;2. 广西民族大学, 广西南宁 530006;3. 广西大学, 广西南宁 530004 |
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| 基金项目： | 广西自然科学基金项目（2018JJA130220）；南宁市科技计划项目（20202091，20192242） |
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| 摘 要： | 【目的】探究组蛋白酶B基因（CtsB）在吉富罗非鱼感染无乳链球菌前后的表达差异，为深入研究罗非鱼抗无乳链球菌感染的免疫应答机制及后续通过SNP分子标记/基因组编辑技术快速获得吉富罗非鱼抗病新品种提供科学依据。【方法】通过RACE克隆罗非鱼CtsB基因cDNA全长序列，使用SMART、 Splign、 MEGA v6.0、 BoxShade及MegAlign等在线软件进行生物信息学及系统进化分析，并以实时荧光定量PCR检测吉富罗非鱼CtsB基因组织表达谱及无乳链球菌感染后CtsB基因的表达变化规律。【结果】吉富罗非鱼CtsB基因cDNA序列全长1746 bp，包括161 bp的5'端非编码区（5'-UTR）、 592 bp的3'端非编码区（3'-UTR）及993 bp的开放阅读框（ORF），共编码331个氨基酸残基。基于CtsB氨基酸序列相似性构建的系统发育进化树显示，吉富罗非鱼与奥利亚罗非鱼的亲缘关系最近，而与鲤鱼、大菱鲆、牙鲆、半滑舌鳎等物种相距较远。CtsB基因在吉富罗非鱼的肝脏、脾脏、鳃、前肾、脑、肠道、皮肤和肌肉等组织中均有表达，且以鳃组织中的相对表达量最高，是其他器官组织的2.0~3.0倍。吉富罗非鱼抗病家系和易感家系感染无乳链球菌后， CtsB基因在脾脏和肾脏中的相对表达量均显著上调 （P<0.05），并于感染后50 h达峰值，且抗病家系的上调表达幅度大于易感家系。【结论】 CtsB基因在鱼类的进化过程中具有高度保守性，但在不同物种中的表达模式存在明显差异。CtsB基因是吉富罗非鱼的重要免疫因子，在抵御无乳链球菌入侵过程中发挥着重要的免疫作用，可作为罗非鱼抗病育种的SNP分子标记给予开发利用。
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| 关 键 词： | 吉富罗非鱼 无乳链球菌 组蛋白酶B基因(CtsB) 表达规律 免疫因子 |
| 收稿时间： | 2021-11-19 |
Cloning of GITF tilapia cathepsin B gene and expression analysis after infected by Streptococcus agalactiae |
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| ZHU Jia-jie,AO Qiu-wei,TAN Yun,LUO Yong-ju,JIANG He-sheng.Cloning of GITF tilapia cathepsin B gene and expression analysis after infected by Streptococcus agalactiae[J].Journal of Southern Agriculture,2022,53(8):2294-2301. |
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| Authors: | ZHU Jia-jie AO Qiu-wei TAN Yun LUO Yong-ju JIANG He-sheng |
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| Affiliation: | 1. Guangxi Academy of Fishery Science, Nanning, Guangxi 530021, China;2. Guangxi Minzu University, Nanning, Guangxi 530006, China;3. Guangxi University, Nanning, Guangxi 530004, China |
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| Abstract: | 【Objective】 To explore expression difference of cathepsin B gene（CtsB）in GITF tilapias before and after infected with Streptococcus agalactiae，so as to provide scientific basis for studying immune response mechanism of tilapia resistant to S.agalactiae and obtaining new disease-resistant GIFT tilapia varieties of by SNP marker or genome editing technique.【Method】 Full-length cDNA sequences of CtsB gene in tilapia were cloned through RACE.Bioinformatics and phylogenetic analysis were conducted through online software such as SMART，Splign，MEGA v6.0，BoxShade and MegAlign.Tissue expression profile of CtsB gene in GIFT tilapia detected by quantitative real-time PCR（qRT-PCR）and change of CtsB gene expression after infected by S.agalactiae.【Result】Full-length cDNA sequences of CtsB gene was 1746 bp，containing a 161 bp 5'untranslated region（5'-UTR），a 592 bp 3'UTR，an open reading frame（ORF）of 993 bp，which encoded a total of 331 amino acid residues.Phylogenetic tree based on CtsB amino acid sequence similarity showed that GIFT tilapia was closely related to Oreochromis aureus，but was far from Cyprinus carpio，Scophthalmus maximus，Paralichthys olivaceus，Cynoglossus semilaevis and other species.The gene was expressed in liver，spleen，gill，kidney，brain，intestine，skin，and muscle of GIFT tilapia，and the highest expression was observed in the gills，which was 2.0-3.0 times higher than that in other organ tissues.After disease-resistant families and susceptible families of GIFT tilapia infected with S.agalactiae，relative expression of CtsB gene in spleen and kidney was up-regulated（P< 0.05），and it peaked after 50 h of infection.Fold of up-regulated expression of disease-resistant families was higher than that of susceptible families.【Conclusion】 CtsB gene is highly conservative in evolution of fish，but expression pattern varies significantly in species.CtsB gene is an important immune factor of GIFT tilapia and it bears important role of immunization against S.agalactiae，so it can be developed and utilized as SNP molecular marker of breeding disease-resistant tilapia.These results suggest that CtsB plays an important role in the immune response to S.agalactiae infection in GIFT tilapia. |
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