黄沙鳖β-防御素基因克隆及其表达分析  

Cloning and expression analysis ofβ-defensin gene of Huangsha turtle

作　　者：许飘尹 杨廷雅 胡大胜 韩书煜 黎姗梅 李明 盛雪晴 梁静真 黄钧 XU Piao-yin;YANG Ting-ya;HU Da-sheng;HAN Shu-yu;LI Shan-mei;LI Ming;SHENG Xue-qing;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;Yuzhou Station of Aquaculture Technology Extension,Yulin,Guangxi 537000,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]广西水产技术推广站,南宁530022 [3]玉州区水产技术推广站,广西玉林537000

出　　处：《南方农业学报》2020年第9期2269-2277,共9页Journal of Southern Agriculture

基　　金：广西自然科学基金项目(2018GXNSFAA138167);广西农业农村厅水产养殖病害测报项目(桂财预函〔2019〕105号)。

摘　　要：【目的】明确β-防御素在黄沙鳖先天免疫中的潜在作用,为开展黄沙鳖绿色病害防治及促进其养殖业健康发展打下基础。【方法】运用RACE克隆黄沙鳖β-防御素基因(Hs-BD1)cDNA序列,采用SignalP-5.0、PredictProtein、PSIPRED、Robetta和Clustal X等在线软件进行生物信息学分析,并通过实时荧光定量PCR检测Hs-BD1基因在黄沙鳖不同组织中的表达特征及细菌感染前后的表达变化。【结果】Hs-BD1基因c DNA序列全长493 bp,包括72 bp的5’非编码区(5’-UTR)、201 bp的开放阅读框(ORF)及220 bp的3’非编码区(3’-UTR)。Hs-BD1基因编码66个氨基酸残基,包括22个氨基酸残基组成的信号肽区、3个氨基酸残基组成的前肽区和41个氨基酸组成的成熟肽区。其中,成熟肽区具有6个保守的半胱氨酸残基(31Cys、58Cys、38Cys、52Cys、42Cys和59Cys)及位于C1和C2间的甘氨酸残基(Gly),即Hs-BD1基因属于β-防御素家族。Hs-BD1氨基酸序列与中华鳖β-防御素16的相似性最高(93.9%),基于β-防御素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和西部锦龟聚为一支,其亲缘关系相对较近。6个保守的半胱氨酸残基分别以C1-C5、C2-C4和C3-C6的连接方式形成3个二硫键;Hs-BD1成熟蛋白三级结构是由α-螺旋、β-折叠和无规则卷曲组成。Hs-BD1基因在黄沙鳖肝脏、脾脏、肺脏和肾脏中的相对表达量较高,在心脏、肠道、肌肉、脑组织和表皮中的相对表达量均较低;以温和气单胞菌攻毒后,Hs-BD1基因在黄沙鳖脾脏中的相对表达量呈上升—下降—上升—下降的变化趋势,在攻毒后第3和36 h共出现2个表达峰值,对应的相对表达量分别是攻毒前(0 h)的20.8和10.6倍,差异均极显著(P<0.01)。【结论】Hs-BD1基因在黄沙鳖的多个组织中均有表达,尤其在肝脏、脾脏、肺脏和肾脏中的相对表达量较高,且可被温和气单胞菌感染诱导表达,说明Hs-BD1基因在黄沙鳖抵�【Objective】This study aimed to clarify the mechanism ofβ-defensin and its potential role in the innate immunity of Huangsha turtle,and lay a foundation for carrying out the green disease prevention and control of Huangsha turtle and promoting the healthy development of its breeding industry.【Method】The cDNA sequence ofβ-defensin gene,HsBD1 in Huangsha turtle was cloned by RACE method,and the bioinformatics analysis was conducted by the online softwares including SignalP-5.0,Predictprotein,PSIPRED,Robetta and Clustal X.The expression patterns in various tissues of Huangsha turtle and the ones after bacterial challenge of Hs-BD1 gene were detected by real-time fluorescence quantitative PCR.【Result】The cDNA sequence of Hs-BD1 gene was 493 bp in length,including a 5’untranslated region(UTR)of 72 bp,an open reading frame(ORF)of 201 bp and a 3’UTR of 220 bp.The Hs-BD1 gene coded 66 amino acids(aa)containing the signal peptide region of 22 aa,the propeptide region of 3 aa and the mature peptide region of 41 aa.The mature peptide had six conserved cysteine(Cys)residues(31Cys,58Cys,38Cys,52Cys,42Cys and59Cys)and the conserved glycine(Gly)residue between C1 and C2,indicating that the amino acid sequence of Hs-BD1 gene belonged to theβ-defensin family.The amino acid sequence of Hs-BD1 shared the highest similarity(93.9%)with Ps-BD16 from Pelodiscus sinensis,and the phylogenetic tree based on the similarity of amino acid sequences ofβ-defensin amino acid also showed that Huangsha turtle,P.sinensis and Chrysemys picta bellii clustered in a clade and their genetic relationships were relatively close.Six conserved cysteine residues formed three disulfide bonds by the conjunction type of C1-C5,C2-C4 and C3-C6.The tertiary structure of mature protein of Hs-BD1 was composed ofα-helix,β-sheet and random coil.The expression levels of Hs-BD1 gene in the liver,spleen,lung and kidney were relatively high and the expression levels were relatively low in the heart,intestine,muscle,brain and skin.The relative express

关 键 词：黄沙鳖 Β-防御素 温和气单胞菌 Hs-BD1基因 表达特征 

分 类 号：S947.1[农业科学—水产养殖]