三角帆蚌AMP基因cDNA全序列的克隆及分子特征研究

Fulllength cDNA cloning and molecular characteristic research of AMP gene in Hyriopsis cumingii

摘要:

以三角帆蚌（Hyriopsis cumingii）为研究材料，根据实验室构建的三角帆蚌cDNA文库中已标注的EST序列, 利用RACE克隆了三角帆蚌AMP基因的cDNA全序列。该cDNA序列全长为970 bp，5′端非翻译区123 bp，3′端非翻译区526 bp，开放阅读框（ORF）长为300 bp，共编码99个氨基酸，包含一段由27个氨基酸组成的信号肽和72个氨基酸的成熟肽，分子量为10 924.7 u。氨基酸序列分析表明，该序列存在明显的跨膜结构和疏水区。同源性分析表明，该基因编码的蛋白与海兔（Aplysia californica）theromacin氨基酸序列的相似度最高为56%，而与贻贝等中发现的抗菌肽defensins、mytilins、myticins和mytimycins相似度较低，推测属于抗菌肽theromacin基因家族。Jpred3软件对三角帆蚌AMP蛋白的二级结构预测结果表明，在第2～22、57～62氨基酸残基处存在α螺旋，因此，三角帆蚌的AMP蛋白是包含跨膜螺旋的膜蛋白。为进一步研究该基因的表达、功能及三角帆蚌病害防御奠定了分子基础。

Abstract:

According to the EST sequence in cDNA library in our lab, the full-length cDNA sequence of AMP gene was cloned in the triangleshell pearl mussel Hyriopsis cumingii by the RACE-PCR. A 970 bp cDNA sequence contained a 123 bp 5′-untranslation region, 526 bp 3′-untranslation region and 300 bp open reading frame(ORF), which encoded 99 amino acids with a signal peptides of 27 amino acids and a mature peptide of 72 amino acids, and molecular mass is 10 924.7 u. Amino acid sequence analysis shows that in the sequence there exist an obvious transmembrane and a hydrophobic region. Homology analysis indicates that this full-length amino acid sequence showed the highest similarity with Aplysia californica theromacin (56%), lower similarity with defensins,mytilins,myticins and mytimycins in mussels, so it is speculated that this gene belongs to theromacin gene families of antibacterial peptides. The prediction results of Jpred3 software for H.cumingii AMP protein secondary structure show that the places of 2-22, 57-62 amino acid residues had the alpha helix, accordingly, AMP in H.cumingii is the membrane protein containng transmembrane spiral. This study may lay the molecular basis for further research on the gene expression, function and disease defense of H.cumingii.