拟穴青蟹PHGPx基因的克隆及其表达分析

Molecular cloning and expression analysis of Sp-PHGPx in Scylla paramamosain

摘要:

为了探讨磷脂氢谷胱甘肽过氧化物酶(phospholipid hydroperoxide glutathione peroxidase,PHGPx)在拟穴青蟹免疫防御反应和精巢发育中的作用,实验从拟穴青蟹转录组数据库中筛选出PHGPx基因的EST序列,利用SMART-RACE 技术克隆得到该基因的全长cDNA,命名为Sp-PHGPx。通过相关生物信息学软件对该序列进行分析,运用实时荧光定量技术对Sp-PHGPx mRNA在成熟雌雄拟穴青蟹各组织、性腺不同发育时期以及H₂O₂和脂多糖(LPS)应激下鳃和肝胰腺组织中的表达情况进行分析。结果发现,Sp-PHGPx基因的cDNA全长为1 024 bp,编码180个氨基酸。同源性分析和系统进化树分析表明,Sp-PHGPx与其他物种的PHGPx(GPx4)亲缘关系较近。实时定量PCR结果显示,Sp-PHGPx在成熟拟穴青蟹雌雄各组织中均有不同程度的表达,其中精巢的表达量最高。在性腺不同发育阶段,Sp-PHGPx在卵巢发育过程中的表达量差异不显著;而在精巢发育过程中,其表达量在精子细胞期(T2期)显著高于精母细胞期(T1期)和成熟精子期(T3期),同时也显著高于卵巢发育各时期的表达量。在LPS应激下,鳃和肝胰腺中Sp-PHGPx的表达量分别在6和12 h显著上调;H₂O₂应激下,鳃中的表达量在3 h显著升高,肝胰腺中的表达量在被检测的各时相中无显著差异。研究表明,Sp-PHGPx可能在免疫防御反应以及精巢的发育和成熟等过程中发挥着重要作用。

关键词:拟穴青蟹;磷脂氢谷胱甘肽过氧化物酶(PHGPx);基因克隆;表达;性腺发育

Abstract:

Phospholipid hydroperoxide glutathione peroxidase(PHGPx)is one of the members of GPx family, which has its unique ability to reduce hydroperoxides in complex lipids such as phospholipid, cholesterol and so on.PHGPx is a key enzyme in the antioxidant defense systems of living organisms, including crustaceans, which plays important roles in the effect of antioxidant and sexual maturity of male.In this study, expressed sequence tag(EST)of Scylla paramamosain PHGPx gene was identified from transcriptome sequencing library and the full length cDNA was cloned by SMART-RACE method, which was named Sp-PHGPx.The full-length cDNA of Sp-PHGPx is 1 024 bp, including a 5'UTR of 140 bp, a 3'UTR of 341 bp and an open reading frame of 543 bp, encoded a duduced protein of 180 amino acids.Sequence comparison and phylogenetic analysis showed that Sp-PHGPx was clustered with PHGPx(GPx4)of other species, far from GPx1 and GPx2.Quantitative real time PCR revealed that Sp-PHGPx was expressed in various tissues of the mature female and male crabs with the highest expression level in testis.During the different stages of gonadal development, there was no significant difference in the development of ovary;while Sp-PHGPx was expressed at the highest level in spermatid stage(T2)and was significantly higher than spermatocyte stage(T1)and mature sperm(T3)during the development of testis.Moreover, the expression levels of T2 are higher than these of other stages of ovary development.When under the challenge of LPS, the expression level of Sp-PHGPx was up-regulated significantly at 6 h in gill and at 12 h in hepatopancrea compared with control.The expression of Sp-PHGPx in gill was induced significantly at 3 h, while there was no significant difference in hepatopancrea when exposed to H₂O₂.Above results suggest that Sp-PHGPx may be involved in immune defense and the male reproduction in S.paramamosain.

Key words:Scylla paramamosain;PHGPx;gene cloning;expression;gonadal development