乌苏里白鲑肝脏表达抗菌肽Leap-2基因的克隆及细菌感染后的组织表达分析  

Cloning and tissue expression of liver-expressed antimicrobial peptide Leap-2 in Ussuri cisco Coregonus ussuriensis exposed to bacterial infection

作　　者：刘恩慧 黄天晴 谷伟[1] 王炳谦[1] 张艳萍 李春雨 徐革锋[1] LIU Enhui;HUANG Tianqing;GU Wei;WANG Bingqian;ZHANG Yanping;LI Chunyu;XU Gefeng(Key Laboratory of Freshwater Aquatic Biotechnology and Breeding, Ministry of Agriculture and Rural Affairs, Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China;Gansu Fisheries Research Institute, Lanzhou 730030, China;Xinjiang Tianyun Organic Agriculture Company Limited, Yili 835708, China)

机构地区：[1]中国水产科学研究院黑龙江水产研究所,农业农村部淡水水产生物技术与遗传育种重点实验室,黑龙江哈尔滨150070 [2]甘肃省水产研究所,甘肃兰州730030 [3]新疆天蕴有机农业有限公司,新疆伊犁835708

出　　处：《大连海洋大学学报》2022年第3期420-427,共8页Journal of Dalian Ocean University

基　　金：中央级公益性科研院所基本科研业务费专项(HSY202103M);财政部和农业农村部:国家现代农业产业技术体系资助(CARS-46);中国水产科学研究院科研业务费项目(2020TD32);农业财政专项“东北地区重点水域渔业资源与环境调查”。

摘　　要：为探究乌苏里白鲑Coregonus ussuriensis Berg肝脏表达抗菌肽Leap-2基因的分子结构和表达特征,采用PCR技术从乌苏里白鲑(体质量为30.0 g±1.2 g)肝脏组织中扩增Leap-2基因的开放阅读框(ORF),使用生物信息学软件对其编码的氨基酸序列进行分析,通过荧光定量PCR(qRT-PCR)检测其在健康组织及嗜水气单胞菌Aeromonas hydrophila、杀鲑气单胞菌Aeromonas salmonicida和金黄色葡萄球菌Staphylococcus aureus感染后的组织表达变化。结果表明:采用PCR方法从乌苏里白鲑肝脏中克隆得到Leap-2基因的ORF区(GenBank:MZ170050),片段长度为276 bp,可编码91个氨基酸,其成熟肽区域包含4个高度保守的半胱氨酸残基;系统进化分析显示,乌苏里白鲑的Leap-2氨基酸序列与银鲑Oncorhynchus kisutch的亲缘关系较近,且具有较高的同源性,一致性为96.7%;乌苏里白鲑肝脏中的Leap-2基因表达量最高,其次为肠和肾,肌肉中的基因表达量最低;3种细菌感染导致Leap-2在各组织中的表达均有所上调,Western blot检测也显示,Leap-2蛋白水平在24 h内表达上调。研究表明,Leap-2基因参与了乌苏里白鲑应对外源干扰的免疫反应,本研究结果可为解析乌苏里白鲑应对病原体的免疫应答机制提供科学参考。To understand the molecular structure and expression characteristics of liver-expressed antimicrobial peptide Leap-2 in Ussuri cisco Coregonus ussuriensis Berg,the open reading frame(ORF)of the Leap-2 gene was amplified from the liver tissue of Ussuri cisco with body weight of(30.0±1.2)g by PCR,and the Leap-2 amino acid sequence was analyze by bioinformatics software.The relative expression levels were detected in healthy tissues and tissues of Ussuri cisco intraperitoneally infected by Aeromonas hydrophila,Aeromonas salmonicida or Staphylococcus aureus at a dose of 100μL/ind.of 1×106 CFU/mL by fluorescence quantitative real-time PCR(qRT-PCR).The results showed that Leap-2 gene(GenBank:MZ170050)had ORF region of 276 bp,which was cloned from the liver of Ussuri cisco by PCR.Leap-2 encoding 91 amino acids and its mature peptide region contained four highly conserved cysteine residues.Phylogenetic analysis showed that Ussuri cisco Leap-2 amino acid sequence was closely related to coho salmon Oncorhynchus kisutch,with the homology of 96.7%.Tissue expression showed that Ussuri cisco Leap-2 was the most abundant in the liver,followed by the intestine and kidney,with the minimal expression level in muscle.The up-regulated expression of Leap-2 was observed in all detected Ussuri cisco exposed to A.hydrophila,A.salmonicida,and S.aureus infection.Western blot revealed that the expression of Leap-2 protein was up-regulated within 24 h,indicating that Leap-2 was involved in the immune response of Ussuri cisco to bacteria.The finding may provide theoretical reference with understanding of the immune response of Ussuri cisco.

关 键 词：乌苏里白鲑 抗菌肽 Leap-2基因 嗜水气单胞菌 杀鲑气单胞菌 金黄色葡萄球菌 荧光定量 

分 类 号：S917.4[农业科学—水产科学] Q786[生物学—分子生物学]