三疣梭子蟹NF-κB家族基因Relish和Dorsal的克隆及表达特征

CLONING AND EXPRESSION OF NF-κB FAMILY GENES IN PORTUNUS TRITUBERCULATUS

为研究Relish和Dorsal在三疣梭子蟹免疫过程中所起到的作用,研究利用RACE技术克隆获得三疣梭子蟹Relish(Pt-Rel)、Dorsal基因(Pt-Dor) cDNA全长,并通过实时荧光定量PCR技术分析了Pt-Rel和Pt-Dor基因在健康蟹不同组织及其原代培养的血淋巴细胞在感染不同微生物后的表达情况。结果显示,Pt-Rel cDNA长3254 bp,ORF长2949 bp,编码983个氨基酸,Pt-Dor cDNA长2348 bp,ORF长1911 bp,编码637个氨基酸;蛋白结构预测分析发现Pt-Rel和Pt-Dor均包含RHD (Rel homology domain)及IPT (Immunoglobulin-like fold,Plexins,TranscriPtion factors)Rel/NF-κB家族蛋白经典结构域。Pt-Rel和Pt-Dor与其他节肢动物Relish、Dorsal氨基酸序列具有很高的相似性;在系统进化分析中Pt-Rel和Pt-Dor分别与中华绒螯蟹等甲壳动物的Relish、Dorsal聚在一支,而昆虫类聚在另一支。Pt-Rel和Pt-Dor在检测的6种组织中均有表达,且2个基因均在血淋巴细胞中表达量最高。蟹血淋巴细胞体外感染实验结果表明,不同病原微生物对2个基因表达的影响非常相似,假丝酵母在2h明显诱导了Pt-Rel和Pt-Dor基因的表达,而金黄色葡萄球菌及溶藻弧菌在感染4h后使Pt-Rel和Pt-Dor基因的表达显著上调。上述研究结果表明Pt-Rel和Pt-Dor基因很有可能参与了三疣梭子蟹的抗感染免疫过程。

Relish and Dorsal,members of nuclear factor-κB(NF-κB)family protein,play crucial role in immune response in Arthropoda.In this study,the cDNA sequences of Relish and Dorsal were cloned by rapid amplification of cDNA ends(RACE).There are 3254 nucleotides in Pt-Rel with a putative open reading frame(ORF)of 2949 bp that encodes a polypeptide of 983 amino acid residues.The Pt-Dor cDNA was 2348 bp in length including an ORF encoding a polypeptide of 637 amino acid residues.Prediction of protein domains revealed that both Pt-Rel and Pt-Dor contained typical NF-κB family structures RHD(Rel homology domain)and IPT(Immunoglobulin-like fold,Plexins,Transcription factors).Alignment of amino acid sequences for Pt-Rel and Pt-Dor revealed high identities with other arthropodas orthologs.Phylogenetic tree analysis based on amino acid sequences showed that the Pt-Rel and Pt-Dor were distinctly clustered with crustaceans Relish and Dorsal,respectively,but separated from insect orthologs.Pt-Rel and Pt-Dor were expressed in all tested tissues with the highest level in hemocytes.Three different pathogenic microorganism significantly induced both Pt-Rel and Pt-Dor expression in primary cultured crab hemocytes at 2h by Candida lusitaniae infection and 4h by Staphylococcus aureus and Vibrio alginolyticus challenge.These data suggest that Pt-Rel and Pt-Dor might be involved in host defense against to microbial pathogens in Portunus trituberculatus.