黄颡鱼PGRP-L基因克隆及其对爱德华氏菌的反应
MOLECULAR CHARACTERIZATION OF A LONG TYPE PEPTIDOGLYCAN RECOGNITION PROTEIN IN YELLOW CATFISH AND ITS RESPONSE TO EDWARDSIELLA ICTALURI
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摘要: 为了研究肽聚糖识别蛋白家族(Peptidoglycan recognition proteins, PGRPs)在黄颡鱼(Pelteobagrusfulvidraco)先天免疫应答中发挥的作用, 根据NCBI中斑马鱼(Danio rerio) 和虹鳟(Oncorhynchus mykiss) PGRP-L的基因信息, 采用简并引物和RACE方法从黄颡鱼肝脏中克隆得到了一个长型PGRP (PfPGRP-L)基因. PfPGRP-L基因的全长cDNA序列大小为1617 bp, 其中5'和3'非翻译区的长度分别为135和72 bp, 开放阅读框为1410 bp, 编码469个氨基酸. 同源性和系统进化分析表明, 黄颡鱼PGRP-L与虹鳟的同源性为60%, 与脊椎动物的PGLYRP2 或PGRP-L聚在一起. 半定量RT-PCR分析发现PfPGRP-L基因在黄颡鱼鳃、胸腺、肝脏、脾脏、肠道、肾脏、头肾、心脏、血液和肌肉组织中均有分布, 但在肠道和脾脏中的表达量较为丰富, 而在肌肉和血液中表达则很少. 用爱德华氏菌刺激后, PfPGRP-L在肝脏、脾脏、肠道及头肾中的表达明显上调. 结果表明, PfPGRP-L在黄颡鱼抵抗病原菌中具有重要作用.Abstract: Peptidoglycan recognition proteins (PGRPs) are a family of pattern recognition receptors (PRRs) of the immune system, exerting the direct antibacterial activity. A long-type PGRP (designated as PfPGRP-L) was identified from yellow catfish, Pelteobagrus fulvidraco. The entire PfPGRP-L cDNA is 1617 bp, including a 135 bp 5'-UTR, a 72 bp 3'-UTR and a 1401 bp open reading frame (ORF). The deduced amino acid sequence of PfPGRP-L with 469 residues showed 60% identity with rainbow trout, Oncorhynchus mykiss PGRP-L. Conserved PGRP domain at the C-terminus was found in PfPGRP-L. Phylogenetic analysis demonstrated that PfPGRP-L was clustered in a same clade with Oncorhynchus mykiss, and formed a long-type PGRP subfamily together with vertebrate PGLYRP2 or PGRP-L members. RT-PCR analysis demonstrated that the mRNA of PfPGRP-L had similar expression patterns, being constitutively expressed in healthy yellow catfish, with the higher expression in intestine and spleen. By real-time PCR analysis, the transcription levels of PfPGRP-L in the liver, spleen, intestine and head kidney were significantly up-regulated after stimulated with Edwardsiella ictaluri. These results suggest that PfPGRP-L is involved in the defensive immune response against bacterial infection.