凡纳滨对虾ATG13基因克隆及其功能分析

Cloning and functional analysis of ATG13 gene of Litopenaeus vannamei

【目的】克隆凡纳滨对虾(Litopenaeus vannamei)自噬相关基因13(ATG13),并分析凡纳滨对虾ATG13基因的组织表达特征及其在外源刺激后的表达变化,为探究ATG13基因在凡纳滨对虾抗病原感染过程中的作用机制提供理论依据。【方法】采用PCR扩增凡纳滨对虾ATG13基因,通过ProtParam、Softberry、SMART、InterProScan等在线软件进行生物信息学分析,并以实时荧光定量PCR检测凡纳滨对虾ATG13基因的组织表达特征及其在哈维氏弧菌(Vibrio harveyi)和Poly(I:C)刺激后的表达变化。【结果】凡纳滨对虾ATG13基因开放阅读框(ORF)为1431 bp,共编码476个氨基酸残基;凡纳滨对虾ATG13蛋白相对分子量为52.4 kD,理论等电点(pI)为5.40,属于疏水性蛋白,不存在信号肽,但其N末端存在1个ATG13功能域(第94~167位氨基酸处)。凡纳滨对虾ATG13氨基酸序列与果蝇(Drosophila melanogaster)ATG13氨基酸序列的相似性高达97%,基于ATG13氨基酸序列相似性构建的系统发育进化树也显示凡纳滨对虾与果蝇的亲缘关系最近。ATG13基因在凡纳滨对虾肝胰腺、肌肉、鳃、中肠、血细胞、心脏、脑和皮肤等组织中均有不同程度的表达,以肝胰腺中的相对表达量最高,其次是肌肉和中肠,在皮肤和血细胞中的相对表达量较低。经哈维氏弧菌和poly(I:C)刺激后,凡纳滨对虾ATG13基因在肝胰腺、中肠和鳃组织中的相对表达量整体上呈先上升后下降的变化趋势,且在刺激后12、24或48 h达最高值,极显著高于对照组(PBS)(P<0.01)。【结论】受哈维氏弧菌或poly(I:C)等外源刺激后,凡纳滨对虾通过上调自噬相关基因ATG13表达以调控机体清除受损的细胞器和维持细胞稳态,即ATG13基因参与凡纳滨对虾抗病原感染的响应过程。

【Objective】This study aimed to clone the autophagy-related gene 13(ATG13)from Litopenaeus vannamei,and investigate its tissue expression pattern and response to exogenous stimulation.These findings provided a theoretical basis for exploring the role of ATG13 gene in the process of disease-resistant infection of L.vannamei.【Method】The ATG13 gene of L.vannamei was amplified by PCR,and bioinformatics analysis was performed by ProtParam,Softberry,SMART and InterProScan online softwares.Real-time fluorescence quantitative PCR was employed to determine the tissue expression of ATG13 gene in L.vannamei and its expression changes after stimulation by Vibrio harveyi and Poly(I:C).【Result】The open reading frame(ORF)of ATG13 gene was 1431 bp,encoding 476 amino acid residues.With a relative molecular weight of 52.4 kD and a theoretical isoelectric pI of 5.40,the ATG13 protein was a hydrophobic protein lacking signal peptide but containing an ATG13 functional domain(94-167 amino acids)at its N-terminus.The ATG13 amino acid sequence of L.vannamei shared a high similarity(97%)with that of Drosophila melanogaster,and the phylogenetic tree based on ATG13 amino acid sequence similarity also indicated the closest relationship between L.vannamei and D.melanogaster.ATG13 gene was expressed in various tissues of L.vannamei,including hepatopancreas,muscles,gills,midgut,blood cells,heart,brain and skin.The relative expression of ATG13 gene was the highest in hepatopancreas,followed by muscle and midgut,and relatively low in skin and blood cells.After stimulation with V.harveyi and Poly(I:C),the relative expression of ATG13 gene in hepatopancreas,midgut and gill tissues of L.vannamei initially increased and then decreased,and reached the highest value at 12,24 or 48 h after stimulation,which was extremely significantly higher than control group(PBS)(P<0.01).【Conclusion】After exogenous stimulation with V.harveyi or Poly(I:C),L.vannamei up-regulates the expression of autophagy-related gene ATG13 to regulate the clearance of damaged organelles and maintain cell homeostasis,suggesting that ATG13 gene is involved in the response process of disease-resistant infection of L.vannamei.