拟穴青蟹Cactus基因的cDNA克隆、序列及生物学功能

Cloning, expression and biological functional characteristics of Cactus from Scylla paramamosain

  • 摘要: 为获取拟穴青蟹Cactus基因cDNA全长、分析基本生物学信息,并初步探索其在病原物刺激下的免疫反应,实验采用RACE技术获得了拟穴青蟹Cactus(SpCactus)基因的cDNA全长序列,其cDNA全长为2 035 bp,开放阅读框(ORF)1 311 bp,编码436个氨基酸,分子量为46.01 ku。对蛋白理化性质进行预测发现,SpCactus为亲水性蛋白,等电点pI为4.91。经预测,SpCactus与其他物种的IκB蛋白具有相似的功能结构域。同源性比对结果显示,SpCactus与凡纳滨对虾和中国明对虾的Cactus蛋白同源性均高达62%,相似度为73%。系统进化树分析显示,SpCactus与甲壳动物聚为一支,与无脊椎动物聚为一大支。实时荧光定量PCR(RT-PCR)检测发现,SpCactus基因在拟穴青蟹不同组织中均有表达,肌肉中的表达量最高,其次为心脏、眼柄、血液和鳃,肝胰腺中的表达量最低。LPS和金黄色葡萄球菌刺激均能显著诱导SpCactus基因的表达。本实验成功扩增了SpCactus基因全长,并进行了生物信息学分析、理化性质预测,初步探讨了其生物学功能,为进一步研究其在免疫反应中的生物学功能提供参考依据。

     

    Abstract: In this study, we obtained the full length of the SpCactus gene by RACE technology from Scylla paramamosain(SpCactus). And we analyzed the biological information of the SpCactus and its immune response under pathogen stimulation. The full length of SpCactus mRNA contains a 5′ untranslated region (UTR) of 228 bp, an open reading frame of 1 311 bp and a 3′ UTR of 496 bp. The SpCactus protein contains five characteristic ANK homology domains and showed 62% identity (73% similarity), 24% identity (36% similarity), and 9% identity (18% similarity) to the Litopenaeus vannamei Cactus (LvCactus) protein, the Drosophila melanogaster Cactus (DmCactus), and the Homo sapiens IκB protein, respectively. Prediction of the protein physicochemical properties of SpCactus revealed that it is a hydrophilic protein. By physical and chemical properties analysis, SpCactus was a hydrophilic protein. And its isoelectric point (pI) is 4.91. The mRNA of SpCactus showed high expression in the muscle, eyestalk and heart, but low expression in the hepatopancreas. Moreover, the expression of SpCactus were significantly upregulated by stimulation with Staphylococcus aureus and lipopolysaccharide. In this study, SpCactus gene was successfully cloned and characterized for the first time, and its bioinformatics analysis, physical and chemical properties were predicted. The biological functions of SpCactus gene were preliminarily explored, providing a basis for further study of its biological functions in immune response.

     

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