异育银鲫PTX3基因的克隆与表达分析

Molecular characterization and expression analysis of Pentraxin gene fromCarassius auratus

为研究Pentraxin 3（PTX3）在鱼类中的作用,克隆得到异育银鲫（Carassius auratus gibelio）PTX3基因,并对其序列结构特征和表达模式进行分析.异育银鲫PTX3序列由3个外显子和2个内含子构成,编码453个氨基酸.异育银鲫PTX3蛋白的前22aa为信号肽,241-453aa为PTX结构域;在PTX结构域有一个非典型的Pentraxin信号.同源进化分析发现,异育银鲫PTX3 与其他鱼类聚为一支,且与斑马鱼最为相似（79%）;两栖类和哺乳类各自聚为一支.实时荧光定量PCR 结果显示,PTX3在异育银鲫的心脏、肝脏、脾脏、头肾、体肾、鳃、脑、肌肉、肠、血液、性腺等11个组织中都有表达.在嗜水气单胞菌攻毒后,肝脏、脾脏、头肾、肠和血液中PTX3mRNA水平都显著上调.在感染后3h,脾脏中PTX3mRNA 水平即出现显著上调;感染后6h,血液中PTX3mRNA水平即达到了峰值.这些结果表明,PTX3作为急相反应蛋白在鱼类受到病原侵袭中发挥免疫保护作用.

We cloned the DNA and cDNA sequence of the Pentraxin 3gene(PTX3)fromCarassius auratus,and examined its expression patterns in various tissues and after Aeromonas hydrophilainfection.Bioinformatics analysis showed that the C.auratus PTX3 gene consists of three exons and two introns,encoding 453 amino acids with the signal peptide(1-22aa)and a pentraxin domain(241-453aa).Sequences analysis revealed that the C.auratus PTX3 amino acids sequence is closest to that of zebrafish(79%).Phylogenetic analysis showed that C.auratus fall in the fish clade.Real-time quantitative PCR results showed that the C.auratus PTX3 is constitutively expressed in the heart,liver,spleen,head kidney,kidney,gills,brain,muscle,intestine,blood,and gonads.The mRNA levels of PTX3 in the liver,spleen,head-kidney,intestine and blood increased significantly after A.hydrophilainfection.At 3h post-infection,the PTX3 mRNA levels in spleen were significantly increased(P<0.05)and at 6hpostinfection,the PTX3 mRNA levels in the blood reached a peak.These results indicated that PTX3 is an acute phase protein which can protect fish against infections.