克氏原螯虾白斑病毒株(WSSV-Cc)基因组的一个印迹  

A footprint in the genome of freshwater crayfish whispovirus(WSSV-Cc)

作　　者：柯飞[1,2] 桂朗 李涛 张奇亚[1,2] KE Fei;GUI Lang;LI Tao;ZHANG Qiya(Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan 430072,China;The Innovation Academy of Seed Design,Chinese Academy of Sciences,Beijing 100101,China;International Research Center for Marine Biosciences,Ministry of Science and Technology,Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 210306,China)

机构地区：[1]中国科学院水生生物研究所,湖北武汉430072 [2]中国科学院种子创新研究院,北京100101 [3]上海海洋大学,国家海洋生物科学国际联合研究中心,水产种质资源发掘与利用教育部重点实验室,上海201306

出　　处：《水产学报》2021年第9期1491-1499,共9页Journal of Fisheries of China

基　　金：国家自然科学基金(31972839,31772890);国家重点研发计划(2018YFD0900601);中国科学院项目(KJZD-SW-L11,XDA24030203);上海市科学技术委员会资助项目(21ZR1427200)。

摘　　要：从自然感染、濒死的克氏原螯虾中分离出的白斑病毒株(Cambarus clarkii whispovirus,WSSV-Cc或Cc株)是一株基因组较小的新毒株。为寻找白斑病毒进化过程在基因组中留下的印迹,进行了显微和超微观察、基因组架构与系统发育分析及相关基因扩增等研究。选择Cc株74L、86L、87R、88R、92R和95R的6个基因,与8个白斑病毒株的同源基因所编码蛋白序列构建进化树,结果可分为克氏原螯虾病毒(Cc株、CN02株、Pc株)和海水对虾病毒(CN株、CN01株、CN03株、CN04株、TW株和KR株)2支。再对不同毒株的同源蛋白进行多重序列比对,显示Cc-87R是与海水对虾病毒株同源蛋白差异显著、缺失跨膜区(TMD)及其相邻287 aa序列、但仍有完整PI3K_bd结构域的病毒膜蛋白。进一步设计和使用87R-F/87R-R和238-F/87R-R两对引物,分别以淡水小龙虾病毒Cc株和海水对虾病毒CN株的基因组为模板进行核酸扩增,结果从Cc株模板中扩增到大小为709 bp,含Cc-87R全部序列的核酸片段;而从CN株的模板中却扩增到大小分别为1600和4810 bp,仅含Cc-87R部分序列的核酸片段,为Cc-87R是Cc株基因组中一个序列结构独特的印迹提供了实验证据。这一发现将有助于克氏原螯虾白斑病毒病原的检测及其流行趋势预警。A white spot syndrome virus strain(Cambarus clarkii whispovirus, WSSV-Cc or Cc strain) was isolated from naturally infected and moribund freshwater crayfish(C. clarkii or Procambarus clarkii, P. clarkii),which is a new strain with a smaller genome. In order to find the footprints in the genome generated during virus evolution, microscopic and ultramicroscopic investigations, genome architecture and phylogenetic analysis, and specific gene amplification were performed. The products of six genes(74 L, 86 L, 87 R, 88 R, 92 R and 95 R) of Cc strain were selected to construct a phylogenetic tree with their homologs from eight white spot virus strains. Results showed that the strains can be divided into two clusters: one included P. clarkii nimavirus(Cc, CN02, and Pc)and the other contained marine shrimp nimaviruses(CN, CN01, CN03, CN04, TW and KR). Multiple sequence alignments of homologous proteins from different viral strains showed that Cc-87 R is significantly different from its homologous membrane proteins of marine shrimp nimavirus. It lacks the transmembrane domain(TMD) and its adjacent 287 aa sequence, but still has a complete PI3 K_rbd region. Further nucleic acid amplification was performed with two pairs of primers, 87 R-F/87 R-R and 238-F/87 R-R, using the genomes of P. clarkii nimavirus Cc strain and marine shrimp nimavirus CN strain as templates respectively. A fragment with size of 709 bp containing the entire sequence of Cc-87 R was amplified from the Cc genomic template. However, the fragments with size of 1 600 and 4 810 bp, only containing partial sequence of Cc-87 R, were amplified from the template of the CN strain. The results provided experimental evidence that Cc-87 R is a footprint with unique sequence structure of the Cc strain. This finding will benefit the detection of the P. clarkii nimavirus pathogen and the warning of its epidemic trend.

关 键 词：克氏原螯虾 克氏原螯虾白斑病毒(WSSV-Cc或Cc株) 基因组 印迹 分子标记 

分 类 号：S941[农业科学—水产养殖]