摘要
【目的】克隆太平洋牡蛎(Crassostrea gigas)新型髓样分化因子88(MyD88)基因MyD88-3,分析其在溶藻弧菌(Vibrio alginolyticus)感染后的表达模式,为探究太平洋牡蛎MyD88基因在免疫调控中的作用和太平洋牡蛎健康养殖提供理论依据。【方法】克隆CgMyD88-3基因编码区(CDS),运用ProtScale、SWISS-MODEL和InterPro等进行生物信息学分析,采用实时荧光定量PCR分析溶藻弧菌感染前后,CgMyD88-3基因在太平洋牡蛎不同组织中的表达情况。【结果】CgMyD88-3基因CDS长度为537 bp,共编码178个氨基酸残基,CgMyD88-3蛋白相对分子量为20.74 kD,理论等电点为6.10。亚细胞定位预测结果显示CgMyD88-3蛋白位于细胞质,含5个酪蛋白激酶II磷酸化位点(17TEED20、29SNLE32、76TQNE79、118TAND121、123TKED126)和3个蛋白激酶C磷酸化位点(26TMK28、148TAK150、169SEK171)。CgMyD88-3氨基酸序列只含1个TIR(Toll/interleukin-1 receptor)结构域,不含Death结构域。CgMyD88-3氨基酸序列与美洲牡蛎(Crassostrea virginica)CvMyD88-like氨基酸序列相似性最高,为100%,基于MyD88氨基酸序列相似性构建的系统发育进化树显示,CgMyD88-3先和美洲牡蛎CvMyD88-like聚为一支,然后与太平洋牡蛎CgMyD88-T1和CgMyD88-T2聚为一支。实时荧光定量PCR检测发现,CgMyD88-3基因在健康太平洋牡蛎6种组织中均有表达,相对表达量最高的为外套膜组织,鳃组织次之,在其他组织中依次为血细胞>性腺>消化腺>闭壳肌。溶藻弧菌感染后,太平洋牡蛎闭壳肌、消化腺、血细胞和鳃组织中的CgMyD88-3基因相对表达量均明显升高,在闭壳肌、消化腺和血细胞中感染72 h后达最高,在鳃中感染6 h后达最高,而在外套膜组织中感染后各时间段的CgMyD88-3基因相对表达量均低于0 h。【结论】CgMyD88-3基因在健康的太平洋牡蛎各组织中均有分布,在外套膜中的相对表达量最高。溶藻弧菌刺激可诱导CgMyD88-3基因高表达,表明CgMyD88-3基因可能在太平洋牡蛎抵御外界微生物及病原体感染中扮演重要角色。
【Objective】The purpose of the study to clone a novel myeloid differentiation factor 88(MyD88)gene MyD88-3 from the Pacific oyster(Crassostrea gigas),the expression pattern of CgMyD88-3 gene was analyzed in different tissues of C.gigas under Vibrio alginolyticus challenge,so as to provide a theoretical basis for exploring the role of MyD88 gene in the immune regulation and the healthy cultivation of C.gigas.【Method】Coding region(CDS)of CgMyD88-3 gene was cloned.The bioinformatics analysis was analyzed by ProtScale,SWISS-MODEL and InterPro.Real-time fluorescence quantitative PCR was used to analyze the expression changes of CgMyD88-3 gene in different tissues of C.gigas.【Result】The CDS length of CgMyD88-3 gene was 537 bp,encoding 178 amino acids residues.The theoretical molecular weight of CgMyD88-3 protein was 20.74 kD and the isoelectric point was 6.10.The sub-cellular localizations of the CgMyD88-3 protein were predicted in the cytoplasm.CgMyD88-3 was predicted to have five casein kinase II phosphorylation sites(17TEED20,29SNLE32,76TQNE79,118TAND121,123TKED126)and three protein kinase C phosphorylation sites(26TMK28,148TAK150,169SEK171).CgMyD88-3 amino acids possessed only TIR(Toll/interleukin-1 receptor)domain but lacked Death domain.CgMyD88-3 amino acid sequence had the highest similarity with CvMyD88-like amino acid sequence of Crassostrea virginica,which was 100%.The result of the phylogenetic tree based on MyD88 amino acid sequence similarity showed that CgMyD88-3 firstly clustered with C.virginica CvMyD88-like,and then with CgMyD88-T1 and CgMyD88-T2 of C.gigas.The real-time fluorescence quantitative RT-PCR detection revealed that CgMyD88-3 was expressed in all six tissues of healthy C.virginica.The highest relative expression was found in the mantle,followed by gills,and in other tissues,the order was haemocytes>gonads>digestive glands,with the lowest relative expression level in the adductor.After infection by Vibrio alginolyticus,the relative expression of CgMyD88-3 was up-regulated in the adductor,digestive glands,haemocytes and gills.The expression of CgMyD88-3 reached peak at 72 h in adductor,digestive glands and haemocytes,respectively,and peaked at 6 h in gills,while the expression of CgMyD88-3 at other times was lower than 0 h after infection in the mantle.【Conclusion】The present results show that CgMyD88-3 is expressed in all tissues of healthy C.virginica,the highest relative expression is found in the mantle.Stimulation by V.alginolyticus obviously induces the up-regulation of CgMyD88-3 expression,indicating that the CgMyD88-3 may play an important role in the defense of C.virginica against external microbial and pathogen infections.
作者
方静
钱敏桦
陈泓霖
杨绍宇
蔡小辉
FANG Jing;QIAN Min-hua;CHEN Hong-lin;YANG Shao-yu;CAI Xiao-hui(College of Marine Sciences,Beibu Gulf University/Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation,Qinzhou,Guangxi 535011,China;Key Laboratory of Environment Change and Resources Use in Beibu Gulf,Ministry of Education,Nanning Normal University,Nanning,Guangxi 530001,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2024年第5期1510-1519,共10页
Journal of Southern Agriculture
基金
国家自然科学基金项目(31272682)
广西北部湾海洋生物多样性养护重点实验室(北部湾大学)自主研发项目(2023ZB08)
北部湾大学海洋科学广西一流学科标志性重大成果基金孵育计划项目(DRC004)
广西高校中青年教师科研基础能力提升项目(2020KY10032)。
