拟穴青蟹两个Crustin新变体的克隆与表达分析

Cloning and expression analysis of two novel Crustin isoforms from Scylla Paramamosain

Crustins是一类广泛存在于甲壳动物中富含半胱氨酸的阳离子抗菌肽.该研究从拟穴青蟹(Scylla Paramamosain)中克隆获得两个Crustin新变体,分别命名为SpCrus1b和SpCrus2b.分析显示两者的前体肽包含N-端信号肽和C-端成熟肽两部分,成熟肽均含有12个位置高度保守的半胱氨酸.SpCrus1b在信号肽和乳清酸蛋白(WAP)结构域之间存在一个半胱氨酸富集区,属于典型的Ⅰ型Crustins;SpCrus2b除具有SpCrus1b的结构特征外,在近N-端额外含有一个甘氨酸富集区,属于典型的Ⅱ型Crustins.基因组结构分析显示SpCrus1b的基因组DNA序列为4个外显子/3个内含子结构,而SpCrus2b的基因组DNA序列为2个外显子/1个内含子结构.两者均主要在鳃中表达,且在脂多糖(LPS)刺激24h和聚肌胞苷酸(PolyI:C)刺激12h后表达量均极显著上调(p<0.01),表明SpCrus1b和SpCrus2b参与了拟穴青蟹抗细菌和抗病毒的先天免疫过程.

Crustins,widely exist in crustaceans,are cationic cysteine-rich antimicrobial peptides(AMPs).In this study,two novel Crustin isoforms were identified from Scylla paramamosain,namelySpCrus1b and SpCrus2b.The analysis showed a signal peptide at the N-terminus and a mature peptide at the C-terminus in both propeptides,and there were 12 cysteine residues at highly conserved positions found in mature peptides.In SpCrus1b,there was a cysteine-rich region(CRR)between the signal peptide and whey acidic protein(WAP)domain,showing a typical character of type ⅠCrustins.Compared with SpCrus1b,SpCrus2b possessed an extra glycine-rich region(GRR)at the N-terminus,which was proposed as type Ⅱ Crustins.In addition,the genomic organization analysis revealed that SpCrus1b had 4 exons/3 introns, while SpCrus2b had 2 exons/1 intron.Both SpCrus1b and SpCrus2b were highly expressed in gills and could be significantly up-regulated(p<0.01)after 24 h lipopolysaccharide(LPS)challenge or 12 h polyinosinic-polycytidylic acid(PolyI:C)challenge. The results suggested that SpCrus1b and SpCrus2b were potential AMPs,which played an important role in the innate immune process of S. paramamosain against bacterial and viral infections.