黄鳝PLA2基因的克隆及表达分析

利用同源克隆技术和RACE技术克隆黄鳝(Monopterus albus)磷脂酶A2(PLA2)基因的cDNA全长;并采用荧光定量RT-PCR法检测PLA2基因在黄鳝各组织中的表达量情况。结果显示:黄鳝PLA2基因全长cDNA大小为2 606 bp(Gen Bank登录号:KX852397),其中开放阅读框2 205 bp,编码736个氨基酸,预测分子大小为83.623 kD,理论等电点5.84;5'和3'非编码区长度分别为208 bp和193 bp。该蛋白序列没有信号肽和跨膜结构。氨基酸序列比对和系统树分析显示,黄鳝PLA2基因的结构十分保守,黄鳝与大黄鱼和金头鲷的PLA2基因亲缘关系最近,与鼠类和蟾蜍的亲缘关系较远。RT-PCR分析表明,PLA2基因在组织中的表达具有组织特异性,在消化器官前肠组织中的表达最高,在性腺及肌肉组织中表达很少。

Cloning and expression analysis of PLA2 from Monopterus albus

In the present study, the rapid-amplification of cDNA ends (RACE) was used to the cDNA sequences of cytosolic phospholipase A2 (PLA2) of Monopterus albus, and the tissue expression analysis was used by RT-PCR in different tissues.The results indicated that the full length cDNA sequence of M.albus PLA2 consist of 2 606 bp, containing an open reading frame (ORF) of 205 bp.It contains a 208 bp 5′ untranslated region (UTR) and a 193 bp 3′ terminal UTR.The putative PLA2 is predicted to be a peptide of 735 amino acids, with a calculated molecular weight of 83.623 kD and an isoelectric point of 5.84.The PLA2 has not predicted signal peptide and transmembrane helices.Amino acid alignment and phylogenetic analyses results showed that the PLA2 amino acid sequence of M.albus was relatively conserved in fish, PLA2 of rice field eel was more closely related to that of Larimichthys crocea and Sparus aurata, but had a distant relationship with rats and toad.The RT-PCR analysis showed that the PLA2 presented the tissue specificity to varying degrees.PLA2 mRNA is constitutively expressed in all the examined organs of healthy rice field eel, with the highest level in foregut of intestinal digestive organs, and with the lower level in gland and muscle.