凡纳滨对虾AP-1基因的克隆和表达特征分析

Cloning andexpression profile analysis of AP-1 gene from Pacific white shrimp(Litopenaeus vannamei)

  • 摘要: 为了探讨凡纳滨对虾转录因子AP-1在病毒引发的免疫应答过程中的潜在作用,实验根据前期的转录组和表达谱结果提示信息,首次克隆了凡纳滨对虾AP-1基因(LvAP-1,GenBank注册号:KF999956),利用在线软件进行了生物信息学分析,运用半定量的方法进行了组织表达分析,并利用实时荧光定量PCR(qPCR)技术分析了该基因在白斑杆状病毒(WSSV)侵染过程中的表达变化特征。结果显示,AP-1基因ORF区全长882 bp,编码293个氨基酸。预测分析显示该基因编码的蛋白质含有1个Jun结构域和1个高度保守的亮氨酸拉链结构域(bZIP),其中Jun结构域在非脊椎动物中保守性不高。组织表达分析表明,该基因在凡纳滨对虾各组织中广泛表达,其中在血细胞中表达量最高。在WSSV感染早期(0.5 hpi),该基因表达没有显著改变,感染后5 h(5 hpi),AP-1基因开始显著上调表达,在人工感染后24 h,该基因的表达量达到最高(P<0.01)。研究表明,该基因在一定程度上参与了凡纳滨对虾体内由WSSV引发的先天免疫应答过程,为进一步研究LvAP-1在对虾应答病毒侵染过程中的功能和作用机制奠定了基础。

     

    Abstract: The purpose is to study Litopenaeus vannamei transcription factor AP-1 potential role in the immune response triggered by the virus.Our previous transcriptome results showed that the expression profile of AP-1 fluctuated significantly in response to WSSV infection.In this research,a novel AP-1 gene(GenBank Accession Number:KF999956)was cloned from Pacific white shrimp(L.vannamei).The full length cDNA of AP-1 has an 882bp ORF encoding 293 amino acids.The predicted molecular weight and calculated isoelectric point of AP-1 was 32.7 KDa and 7.80.The online software(SMART)analysis results showed that two predicted domains including the Jun protein domain and a highly conserved basic leucine zipper(bZIP)which existed in the deduced protein of AP-1.Multiple alignment analysis shows that AP-1 shared 48%,42% and 43% amino acid identities with Ixodes Scapularis,Mus musculus and Homo sapiens,respectively.Tissue expression analysis by Semi-quantitative RT-PCR revealed that expression of AP-1 gene is constitutive in all selected tissues and shows highest expression in hemocytes.The transcript of AP-1 is induced in response to white spot syndrome virus(WSSV)infection.At the early stage of WSSV infection(0.5 hpi),AP-1 did not change significantly.Then the expression of AP-1 gene began to increase after 5 hpi.At 24 hpi,the gene expression increased significantly and reached the highest level(P<0.01).From this result,it could be deduced that AP-1 gene took part in shrimp innate immune response caused by WSSV.

     

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