| 摘要: |
| 采用构建三疣梭子蟹血细胞全长cDNA 文库的方法克隆了一个新型抗菌肽基因Pthyastatin,并用荧光定量RT-PCR 方法, 进行Pthyastatin 的表达研究。结果表明, Pthyastatin 前体由16 个氨基酸残基的信号肽和成熟肽两部分组成, 其中Pthyastatin 成熟肽有两个结构域：N 端的富含Pro/Arg 结构域和C 端的包含6 个保守Cys 残基与对虾抗菌肽penaeidins 同源的结构域, 表明Pthyastatin 属于对虾抗菌肽penaeidins 家族; 荧光定量RT-PCR 分析结果表明Pthyastatin 在血细胞中高水平组成型表达;致病菌副溶血弧菌诱导后Pthyastatin 在血细胞中表达先下调、后上调, 在诱导后24h 表达量又基本
恢复到诱导前水平, 支持Pthyastatin 在血细胞中高水平组成型表达的观点, 但致病菌诱导后表达变化机制不明。
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| 关键词: 三疣梭子蟹, 血细胞, 抗菌肽, Pthyastatin, mRNA 表达 |
| DOI：10.11693/hyhz201002013013 |
| 分类号: |
| 基金项目:国家科技支撑计划项目, 2007BAD43B08 号; 浙江省重大科技专项重大项目, 2007C02001 号; 浙江省面上项目,2009C32019 号; 浙江省教育厅项目, 20061141 号 |
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| cDNA CLONING, CHARACTERIZATION AND mRNA EXPRESSION OF A HYASTATIN-LIKE GENE FROM SWIMMING CRAB PORTUNUS TRITUBERCULATUS |
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SHEN Wang, YE Mao, SHI Ge, WANG Ri-Xin
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Laboratory of Marine Biology Resources and Molecular Engineering, Ocean Science College, Zhejiang Ocean University
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| Abstract: |
| Pathogenic challenges in decapod crustaceans are combated by innate immune responses, including the production and secretion of soluble antibacterial peptides into hemolymph. Hyastatin antimicrobial homologues, containing a domain of six Cys residues at the C-terminus, had been identified from the haemocyte library of the swimming crab, Portunus trituberculatus and designated Pthyastatin. Sequence analysis of these cDNAs indicated the presence of five isoforms of Pthyastatin. The full-length cDNA of Pthyastatins contain an open reading frame of 381 or 387bp encoding a precursor of 127 or 129 amino acids that comprise 16 amino acid signal peptides and 111 or 113 amino acid mature peptides. The putative mature peptides consist of two distinctly different domains: a Pro/Arg-rich region at the amino-terminus and a C-terminal region containing six Cys residues with a Cys pattern resembling the one found in penaeidins. Seqences alignment with Hyas araneus Hyastatin and Pthyastatins indicated the signal petide domain and six-Cys domain conserved, but Pthyastatin lacked of a Gly-rich domain which is in the N-terminal region of the mature H. araneus Hyastatin, insteaded of an extended Pro/Arg-rich domain. It suggests that Pthyastatin is a new member of the antimicrobial peptide group containing a six-Cys-residue domain at the C-terminus. Quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay were developed to assess the mRNA expression of Pthyastatin in different tissues and the temporal expression of Pthyastatin in the hemocytes challenged by pathogen, Vibrio Parahaemolyticus. The results indicate that Pthyastatin transcript is constitutively expressed and mainly found in hemocytes. But the expression of Pthyastatin in the hemocytes was enigmatic, down-regulated in the initial hours and up-regulated subsequently and returned to unstimulated level about 24 h, after stimulated by V. parahaemolyticus.
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| Key words: Portunus trituberculatus, Hemocyte, Antimicrobial peptide, Pthyastatin, mRNA expression |
