仿刺参硫酸软骨素合酶1基因克隆及灿烂弧菌感染后的表达特征模式分析  

Cloning of the chondroitin sulfate synthase 1 gene and its expression pattern analysis under Vibrio splendidus infection in sea cucumber Apostichopus japonicus

作　　者：谭颜廷 葛建龙 廖梅杰[2,3] 荣小军 王锦锦[2,3] 李彬 赵岩峰 王印庚[2,3] 王璐 TAN Yanting;GE Jianlong;LIAO Meijie;RONG Xiaojun;WANG Jinjin;LI Bin;ZHAO Yanfeng;WANG Yingeng;WANG Lu(Fisheries College,Ocean University of China,Qingdao 266003,China;Key Laboratory of Sustainable and Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Laboratory for Marine Fisheries Science and Food Production Process Laoshan Laboratory,Qingdao 266071,China;Qingdao Marine Management and Support Center,Qingdao 266071,China)

机构地区：[1]中国海洋大学水产学院,山东青岛266003 [2]中国水产科学研究院黄海水产研究所,农业农村部海洋渔业可持续发展重点实验室,山东青岛266071 [3]崂山实验室海洋渔业科学与食物产出过程功能实验室,山东青岛266071 [4]青岛市海洋管理保障中心,山东青岛266071

出　　处：《中国水产科学》2024年第4期488-499,共12页Journal of Fishery Sciences of China

基　　金：国家重点研发计划项目(2022YFD2400105);山东省重点研发计划项目(2023LZGC019);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金项目(2023TD29)。

摘　　要：硫酸软骨素在生物体应对病毒和细菌感染中起着重要作用,为研究硫酸软骨素合酶1(chondroitinsulfate synthase-1,ChSy-1)在仿刺参(Apostichopus japonicus)受到灿烂弧菌(Vibrio splendidus)感染后的作用,本研究开展了仿刺参ChSy-1基因(AjChSy-1)全长序列克隆和结构及系统进化分析,并采用荧光定量PCR技术测定了该基因在刺参不同组织中的表达差异及灿烂弧菌感染下的表达模式。结果显示,AjChSy-1基因cDNA全长为2756bp,其中,5′-UTR长度为194 bp,3′-UTR为228 bp,ORF为2334 bp,编码777个氨基酸。AjChSy-1基因编码的蛋白含有糖基转移酶保守性DXD基序、β3-糖基转移酶基序及β4-糖基转移酶基序等结构。刺参基因组比对发现在chr13染色体上存在2个AjChSy-1基因拷贝。系统进化分析表明,刺参AjChSy-1编码蛋白与玉足海参(Holothurialeucospilota)和棘冠海星(Acanthaster planci)的蛋白亲缘关系较近,与斑马鱼(Danio rerio)、智人(Homo sapiens)等脊椎动物的蛋白亲缘关系较远。荧光定量PCR显示,AjChSy-1基因具有广泛的组织表达特性,其中,体腔细胞中表达量最高,体壁、性腺(雄性和雌性)和纵肌次之。在响应灿烂弧菌侵染过程中,随灿烂弧菌侵染时间增加,体壁中AjChSy-1表达量呈现先上升后下降的趋势,在第3、6、9天分别为对照组的1.79、2.06、3.12倍,对发病期抗病群体和易感群体中该基因的检测结果表明,易感群体体壁该基因表达量显著低于抗病群体(P<0.05),表达量降低11.4%。推测该基因可能在刺参应对灿烂弧菌中发挥免疫防御作用,相关研究结果为解析刺参AjChSy-1基因功能以及抗病分子调控机制提供了参考数据。Chondroitin sulfate synthase-1(ChSy-1) is a crucial enzyme involved in the synthesis and elongation of chondroitin sulfate(CS),playing a vital role in combating bacterial and viral infections.The growing scale of aquaculture has led to the emergence of "rotting skin syndrome" caused mainly by Vibrio splendidus,posing a severe threat to the healthy cultivation of sea cucumbers.Our recent transcriptome study has found that ChSy-1may have an essential immune function in protecting sea cucumbers from pathogenic infection;however,there has been no prior research on chondroitin sulfate synthase in echinoderms.In the present study,we obtained the full-length cDNA sequence of ChSy-1 using the RACE technique and analyzed its tissue expression characteristics in sea cucumbers using real-time fluorescence quantitative PCR.Furthermore,we also examined the expression characteristics of the body wall tissues under Vibrio vulnificus infiltration to investigate the possible physiological effects of AjChSy-1 on pathogenic infection.The study found that the AjChSy-1 gene has a full cDNA length of 2756 bp,with 5′-UTR length of 194 bp,3′-UTR length of 228 bp,and ORF length of 2334 bp.The ORF encodes 777 amino acids,with a predicted molecular weight of the encoded protein of 89.1 kDa.The theoretical isoelectric point was 7.62,with a fat coefficient of 82.25 and an instability factor of 43.77.The protein sequence contains a type II transmembrane topology with 11 cysteine residues and four potential N-glycosylation sites.The three-dimensional spatial structure of the sea cucumbers AjChSy-1 protein is similar to that of the Holothuria leucospilota ChSy-1,with some differences from Homo sapiens.The sea cucumbers genome contains two copies of the AjChSy-1 gene,both of which are found on chromosome chr13.Gene structure analysis shows that both copies have two exons and one intron.Phylogenetic analysis showed that the protein encoded by AjChSy-1 in sea cucumbers was closely related to that of Holothuria leucospilota and Acanthaster plan

关 键 词：仿刺参 ChSy-1 基因克隆 荧光定量PCR 基因表达 灿烂弧菌 

分 类 号：S947[农业科学—水产养殖]