斑节对虾Chitinase-2基因的克隆及其在蜕皮和幼体发育过程中的表达分析  

Cloning and expression analysis of Chitinase-2 from Penaeus monodon during molting cycle and different larval developmental stages

作　　者：周凯敏[1,2] 江世贵[1] 黄建华[1] 杨其彬[1] 姜松[1] 邱丽华[1] 杨丽诗[1] 周发林[1] 

机构地区：[1]中国水产科学研究院南海水产研究所农业部南海渔业资源开发利用重点实验室,广东广州510300 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《南方水产科学》2017年第4期59-68,共10页South China Fisheries Science

基　　金：现代农业产业技术体系建设专项资金(CARS-47);广东省省级科技计划项目(2014B020202003);国家高技术研究发展计划(863计划)项目(2012AA10A409);广东省海洋与渔业科技推广专项(A201501A06);深圳市生物产业发展专项资金现代农业生物产业推广扶持计划项目(NYSW201400331010053);海南省应用技术研法与示范推广专项(ZDXM2015056)

摘　　要：研究以斑节对虾(Penaeus monodon)转录组获得的几丁质酶基因(Pm Chi)片段,运用RACE技术克隆了PmChi基因c DNA全长,命名为PmChi-2。PmChi-2基因c DNA全长2 050 bp,其中5'-非编码区(5'-UTR)144 bp、3'-UTR 319 bp和开放阅读框(ORF)1 587 bp,编码528个氨基酸。生物信息学分析显示,PmChi-2与其他甲壳动物Chi-2的相似性为78%~97%。实时荧光定量PCR结果显示,PmChi-2在蜕皮前期(D期)表皮中表达水平最高,在胃、鳃、腹神经节和眼柄中表达水平依次降低,在其他组织(肝胰腺、肠、肌肉、心脏)中几乎不表达。不同蜕皮阶段,PmChi-2在鳃、胃和表皮3种组织中的表达变化模式基本一致,均在蜕皮期(E期)表达量最低,而最高表达量在鳃中出现在蜕皮后期(A期),在胃和表皮中为D期。幼体不同发育阶段分析揭示PmChi-2 mRNA在幼体发育阶段的无节幼体到糠虾幼体第二期表达量维持在一个低水平,在糠虾幼体第三期PmChi-2 mRNA表达水平显著升高,在仔虾期又显著下降,推测PmChi-2 mRNA可能与斑节对虾幼体发育密切相关。研究结果表明PmChi-2基因可能在斑节对虾蜕皮以及幼体的变态发育中发挥重要作用,为深入研究斑节对虾几丁质酶发育调控提供了重要信息。We obtained the full-length cDNA sequence of PmChi （GenBank accession number： KX610684, namely PmChi-2） from black tiger shrimps （Penaeus monodon） by transeriptome sequencing and rapid amplification of cDNA ends （RACE）. The full length of PmChi-2 cDNA was 2 050 bp, including a 1 587 bp ORF （open reading frame） which encoded 528 amino aeid residues, a 144 bp 5＇-UTR and a 319 bp 3＇-UTR. Phylogenetic analyses suggest that the predicted amino acid sequence of PmChi-2 shared 78% -97% identity with the type of Chi-2 of other crustaceans. Relative gene expression levels of PmChi-2 gene in various tissues were then detected by qRT-PCR （ quantitative real-time PCR） during the molting cycle and different larval developmental stages. The results show that PmChi-2 had the highest gene expression level in cuticle in the premolt, then decreased in a descending order （ stomach 〉 gill 〉 thoracic ganglia 〉 eyestalk）, while weak expression was detected in the other tissues. The gene expression patterns of PmChi-2 were different among tissues during the molting cycle. PmChi-2 mRNA had the highest expression level at Stage A and the lowest expression level at Stage E in gill. PmChi-2 mRNA had the highest expression level at Stage D and the lowest expression level at Stage E in stomach. PmChi-2 mRNA had the highest expression level at Stage D and the lowest expression level at Stage E in cuticle, which suggests that PmChi-2 may participate in the molting. At different larval developmental stages, PmChi-2 mRNA had the highest expression level at Mysis 3, indicating that PmChi-2 was associated with larval metamorphosis. The results lay a foundation for research on the biological function and regulation of chitinase in P. monodon.

关 键 词：斑节对虾 几丁质酶 基因克隆 表达分析 蜕皮 变态 

分 类 号：S917.4[农业科学—水产科学]