海蜇(Rhopilema esculentum)磷脂酶A_2基因的cDNA、基因组克隆与表达分析

cDNA, Genome Cloning, and mRNA Expression of Phospholipase A_2 Gene from the Rhopilema Esculentum

本研究利用RACE和RT-PCR技术克隆了海蜇(Rhopilema esculentum)磷脂酶A_2基因(Re-PLA_2-1)的cDNA及基因组序列,并分析了其m RNA在海蜇不同发育阶段的表达。Re-PLA_2-1基因的cDNA全长为824 bp,包括了48 bp的5?非编码区、504 bp的开放阅读框及272 bp的3'非翻译区。SMART分析显示,Re-PLA_2-1为分泌蛋白,包括了一个由19个氨基酸组成的信号肽和一个由118个氨基酸组成的磷脂酶A_2结构域。多序列比对和系统进化分析显示,Re-PLA_2-1基因与来自星状海葵(Nematostella vectensis)、僧袍芋螺(Conus magus)、长牡蛎(Crassostrea gigas)等磷脂酶A_2的相似性较高,共同聚类为pfam09056 GIX PLA_2分支,均包含pfam09056家族成员酶活性所必需的Ca^(2+)结合位点、活性催化位点和PLA_2结构域所必需形成二硫键的半胱氨酸。Re-PLA_2-1基因组全长为2671 bp,由4个外显子和3个内含子组成。RT-PCR结果显示,Re-PLA_2-1基因在海蜇4个发育阶段均有表达,其中,横裂体阶段的表达量最高,碟状体阶段最低。这些研究结果为进一步了解海蜇磷脂酶A_2毒素的生物功能奠定了基础。

The cDNA and gene of phospholipase A2 （Re-PLA2-1） of Rhopilema esculentum were cloned using RACE, and the mRNA expression was monitored at different developmental stages with quantitative real-time PCR analysis. The full-length cDNA of Re-PLA2-1 was 824 bp, containing a 5＇-untranslated region （5＇-UTR） of 48 bp, an open reading frame （ORF） of 504 bp, and a 3＇- untranslated region （3＇-UTR） of 272 bp. SMART analysis showed that Re-PLA2-1 was a secreted protein, including a putative signal peptide consisting of 19 amino acid residues and a domain of phospholipase A2. The deduced amino acid sequence of Re-PLA2-1 was highly similar to those of PLA2s from Conus magus, Nematostella vectensis, Crassostrea gigas and so on, and they could form a cluster of pfam09056 GIX PLA2 revealed by the multiple sequence alignment and phylogenetic analysis. They shared the essential features of pfam09056 PLA2s family, including a calcium-binding site, the catalytic active sites, and a PLA2 domain, which perfectly corresponds to the conserved disulfide-bonded cysteine residues involved in the formation of the internal disulfide. The size of Re-PLA2-1 gene was 2671 bp that included four exons and three introns. Quantitative real-time PCR analysis revealed that the expression of Re-PLAz-1 mRNA occurred in all four developmental stages. The expression was the highest in strobila and the lowest in ephyra. These results contributed to further understanding the biological function of PLA2 in R. eseulentum.