黄沙鳖抗菌肽cathelicidin基因克隆及表达特征分析  

Cloning and expression profile of cathelicidin gene of Huangsha turtle

作　　者：马沙 盛雪晴 许飘尹 贺晓晨 黎姗梅 韦汉英 韦慕兰 黄立春 梁静真 黄钧 MA Sha;SHENG Xue-qing;XU Piao-yin;HE Xiao-chen;LI Shan-mei;WEI Hanying;WEI Mu-lan;HUANG Li-chun;LIANG Jing-zhen;HUANG Jun(Institute of Animal Science and Technology,Guangxi University/Guangxi Aquatic Animal Disease Diagnostic Laboratory,Nanning 530005,China;Guangxi General Station of Aquaculture Technology Extension,Nanning 530022,China;Dahua Station of Aquaculture Management,Dahua,Guangxi 530800,China;Aquaculture Technology Extension Station of Du’an,Du’an,Guangxi 530700,China;Nanning Station of Aquatic Housbandry and Veterinary Technology Extension,Nanning 530001,China)

机构地区：[1]广西大学动物科学技术学院/广西水生动物病害诊断实验室,南宁530005 [2]广西水产技术推广总站,南宁530022 [3]大化县水产管理站,广西大化530800 [4]都安县水产技术推广站,广西都安530700 [5]南宁市水产畜牧兽医技术推广站,南宁530001

出　　处：《南方农业学报》2019年第7期1605-1612,共8页Journal of Southern Agriculture

基　　金：广西自然科学基金项目（2015GXNSFBA139063,2018GXNSFAA138167）;广西水产畜牧兽医局水产养殖动物病情测报专项（桂渔牧财〔2017〕23号）

摘　　要：【目的】克隆黄沙鳖cathelicidin基因并分析其组织分布特征及在攻毒后的表达变化,为深入研究cathelicidin功能及在黄沙鳖先天免疫中的潜在作用提供基础数据。【方法】采用RT-PCR结合RACE克隆黄沙鳖cathelicidin基因cDNA全长序列,利用生物信息学分析软件对黄沙鳖cathelicidin基因及其推导氨基酸序列进行结构特征分析,并采用实时荧光定量PCR检测黄沙鳖cathelicidin基因在不同组织中的表达特征及攻毒后的表达变化。【结果】黄沙鳖cathelicidin基因cDNA序列全长1026 bp(GenBank登录号MK250818),包括483 bp的开放阅读框(ORF)、315 bp的5'非编码区(5'UTR)和228 bp的3'非编码区(3'UTR)。黄沙鳖cathelicidin基因cDNA序列编码160个氨基酸,包括氨基端的信号肽区域、含有4个保守半胱氨酸(Cys)残基的cathelin区域及羧基端的成熟肽区域。黄沙鳖cathelicidin基因推导氨基酸序列与中华鳖cathelicidin基因推导氨基酸序列(KY948708.1)的相似性最高,为97.5%;基于cathelicidin基因推导氨基酸序列相似性构建的系统发育进化树也显示黄沙鳖与中华鳖的亲缘关系最近。黄沙鳖cathelicidin基因在脾脏和肝脏中的相对表达量相对较高,其次是心脏,在脑组织、肾脏、肠道、肌肉、表皮和背甲的相对表达量较低。以温和气单胞菌攻毒后,黄沙鳖脾脏cathelicidin基因相对表达量呈升—降—升—降的波动变化趋势,出现2个峰值(攻毒后第3和36 h),对应的cathelicidin基因相对表达量分别是对照(注射等量无菌生理盐水)的23.1和3.5倍。【结论】黄沙鳖cathelicidin基因在脾脏和肝脏中高表达量,且可被温和气单胞菌感染诱导,说明cathelicidin基因在黄沙鳖抵抗病原感染过程中发挥重要作用。【Objective】The antimicrobial peptide cathelicidin gene of Huangsha turtle was cloned,and its tissue expression pattern as well as the expression variation after bacterial challenge were analyzed in order to give basic data for researching the function and the potential role of cathelicidin gene in the innate immune of Huangsha turtle.【Method】The full length sequence of cDNA of Huangsha turtle cathelicidin gene was cloned by RT-PCR along with RACE method.The structure characteristics of cathelicidin gene and its deduced amino acid sequence were analyzed by using bioinformatics analysis software.The expression profile of cathelicidin in different tissues and expression change after bacterial challenge were measured by real-time fluorescence quantitative PCR.【Result】The full cDNA sequence of Huangsha turtle cathelicidin gene was 1026 bp(GenBank accession number:MK250818)in length containing an open reading frame(ORF)of 483 bp,a 5'untranslated region(UTR)of 315 bp and a 3'UTR of 228 bp.The cDNA sequence of Huangsha turtle cathelicidin gene coded 160 amino acids which had a signal peptide region,a cathelin region with 4 conserved cysteine(Cys)residues and a carboxy-terminal mature peptide region.The deduced amino acids sequence of Huangsha turtle cathelicidin gene had the highest similarity(97.5%)with that of Pelodiscus sinensis(KY948708.1).The phylogenetic tree based on the similarity of deduced amino acid sequences of cathelicidin gene also showed the closest relationship between Huangsha turtle and P.sinensis.The expression levels of Huangsha turtle cathelicidin gene in the spleen and liver were relatively high followed by the heart tissue.And its relative expression levels were low in the brain,kidney,intestine,muscle,skin and tergum tissues.The relative expression levels of cathelicidin gene in the spleen of Huangsha turtle showed an upwarddownward-upward-downward trend post injection of Aeromonas sobria.There were two peak values(at 3 and 36 h post injection)in the relative expression levels of cathe

关 键 词：黄沙鳖 抗菌肽 CATHELICIDIN 基因克隆 表达特征 

分 类 号：S966.5[农业科学—水产养殖]