凡纳滨对虾Arf6基因的克隆及表达分析

Molecular cloning and expression analysis of Arf6 in Litopenaeus vannamei

中文摘要:

      通过电子克隆所得序列设计引物，采用PCR扩增方法首次克隆得到长695bp的凡纳滨对虾Arf6基因序列，其中包括96bp的5′非翻译区（Untranslated region, UTR）、71bp的3′ 非翻译区和528bp的开放阅读框。编码175个氨基酸残基，推算分子量约为20kDa，理论等电点为8.82，分子式为C896 H1426 N252 O255 S8。氨基酸序列分析表明， Arf6基因编码的氨基酸序列中具有保守的GTP结合区域，总的带负电荷的残基(Asp+Glu)为21，总的带正电荷的残基(Arg Lys)为25。与其他物种的Arf6基因进行同源比对发现，该基因的氨基酸序列具有较高的保守性，基于氨基酸序列的聚类分析表明，凡纳滨对虾与果蝇属种类亲缘关系最近。半定量RT PCR的结果显示，该基因在组织中广泛表达且在肝胰腺中表达量最高，在眼柄中表达最低；其在经桃拉综合征病毒(TSV)感染后的凡纳滨对虾肝胰腺中的表达量显著高于在无特定病原(SPF)凡纳滨对虾；加之ProtFun在线预测蛋白质的功能分类，表明Arf6基因可能是一免疫应答因子参与凡纳滨对虾免疫防御反应。

英文摘要:

      According to the sequence from the electronic cloning, a pair of primers were designed to clone the Arf6 gene in Litopenaeus vannamei by PCR. A 695bp cDNA was obtained, which contained a 96bp 5′ untranslated region (UTR), a 528bp open reading frame (ORF), and a 71bp 3′ UTR. The encoded protein was composed of 175 amino acids, with a molecular weight of 20kDa and the calculated isoelectric point of 8.82. The molecular formula was predicted to be C896 H1426 N252 O255 S8. The deduced amino acid sequence showed that the protein encoded by the Arf6 gene owned a conserved GTP-binding domain, 21 negatively charged residues (Asp+Glu) and 25 positively charged residues (Arg Lys). The obtained sequence was compared with the other species through BLAST analysis, indicating that the amino acid sequence cloned in this study is highly conserved. Phylogenetic analysis based on the amino acid sequences revealed that L. vannamei is closely related to Drosophila melanogaster. Semi-quantitative RT-PCR results showed that the gene was expressed in most of the examined tissues, and the highest expression level was observed in the hepatopancreas, while the lowest in the eyestalk. Arf6 gene expression level in the hepatopancreas of L. vanname that was infected by the taura syndrome virus (TSV) was higher than that of the specific pathogen free L. vanname. The protein functional classification results suggested that the Arf6 plays a potential role in the pathogen host defense systems as an immune response factor.