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

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.