三疣梭子蟹JHEH基因的克隆及表达分析

Cloning and Expression Analysis of JHEH Gene in Portunus trituberculatus

为了探究JHEH在甲壳动物中的作用,利用RACE技术克隆获得了三疣梭子蟹JHEH(PtJHEH)基因的全长cDNA序列,运用生物信息学分析了JHEH的分子特征,并通过实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)方法检测了JHEH基因在三疣梭子蟹不同组织、蜕皮周期和卵巢发育时期中的表达水平变化。PtJHEH全长1803 bp,包含1374 bp的开放阅读框(ORF),编码467个氨基酸。推导的氨基酸序列具有跨膜结构域和信号肽,包含典型的环氧化酶催化结构域。qPCR结果显示,PtJHEH转录本在各个组织中均有表达,但在肝胰腺中表达量最高,显著高于其他组织(P<0.05),这可能与肝胰腺是甲壳动物物质代谢的主要场所有关。在蜕皮周期中,肝胰腺PtJHEH在蜕皮前期D2期表达量最大,而Y器中PtJHEH则在蜕皮后期表达水平较高,随后显著下降。在卵巢发育过程中,肝胰腺和Y器PtJHEH的表达水平均在卵黄合成晚期(Ⅲ期)升至最高。PtJHEH在蜕皮周期和卵巢发育中的表达模式与课题组已报道的血淋巴MF水平存在一定相关性,推测其参与MF的降解代谢过程。

To explore the role of JHEH in crustaceans,the full-length cDNA of JHEH from the swimming crab,Portunus trituberculatus(PtJHEH)was cloned and characterized using the RACE technique followed by bioinformatics analysis,and its gene expression levels were detected in different tissues,molting cycles and ovarian development stages using the Real-time quantitative PCR(RT-qPCR).The obtained PtJHEH was 1803 bp in length,containing an open reading frame(ORF)of 1374 bp,which encoded 467 amino acids.The deduced amino acid sequence was predicted to have transmembrane domains,signal peptides,and typical epoxide catalytic domain.The qPCR results showed that PtJHEH was expressed extensively in various tissues,but the highest mRNA level was found in the hepatopancreas,which was significantly higher than other tissues(P<0.05).This might be the reflection that the hepatopancreas was the main site of crustacean metabolism.During the molt cycle,the PtJHEH in the hepatopancreas expressed at highest level in the pre-molt D2 stage,while its expression in Y-organs was remarkably high at the post-molt stages(A and B stages),but then decreased significantly.During the ovarian development process,the expression of PtJHEH in hepatopancreas and Y-organs both increased to the highest level at the late-vitellogenic stage(stageⅢ).The expression pattern of PtJHEH in the molting cycle and ovarian development showed a correlation to the fluctuation in the hemolymph MF titers previously reported by our group.This indicates that JHEH may have a role in the regulation of MF degradation.