Molthathong Sudkhate, Rojtinnakorn Jiraporn, Senapin Saengchan, Flegel Timothy W
Center of Excellence for Shrimp Molecular Biology and Biotechnology (Centex Shrimp), Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Fish Shellfish Immunol. 2007 Jun;22(6):617-27. doi: 10.1016/j.fsi.2006.08.008. Epub 2006 Aug 17.
Nucleases are phosphodiesterases that hydrolyze DNA and/or RNA. In a search for shrimp nucleases involved in apoptosis, we discovered a nuclease from hepatopancreatic cDNA of the black tiger shrimp Penaeus monodon. The full-length nuclease gene was amplified and revealed to contain 1668bp corresponding to 381 deduced amino acid residues in the mature enzyme. Sequence analysis indicated 83% nucleic acid identity and 89% amino acid identity to a nuclease from the Kuruma shrimp Penaeus japonicus (also called Marsupenaeus japonicus). Comparative analysis of sequences, conserved motifs and phylogenetic trees indicated that P. monodon nuclease (PMN) belonged to the family of DNA/RNA non-specific endonucleases (DRNSN). RT-PCR analysis using primers specific for PMN mRNA with seven different shrimp tissues revealed that expression in normal shrimp was restricted to the hepatopancreas. Semiquantitative RT-PCR analysis of PMN using hepatopancreatic mRNA from normal shrimp and from shrimp challenged with white spot syndrome virus (WSSV) indicated significant up-regulation of PMN in the hepatopancreas (P<0.05) at the early stage of viral infection but a return to baseline levels as gross signs of disease developed. At the same time, expression was always confined to the hepatopancreas and never seen in other tissues, including those reported to be prime targets for WSSV and subject to increased levels of apoptosis after infection. The results suggested that PMN is probably a digestive enzyme that is unlikely to be involved in hallmark DNA digestion associated with apoptosis.
核酸酶是一类能水解DNA和/或RNA的磷酸二酯酶。在寻找参与对虾凋亡过程的核酸酶时，我们从黑虎虾（斑节对虾）的肝胰腺cDNA中发现了一种核酸酶。该核酸酶全长基因被扩增出来，发现其含有1668bp，对应成熟酶中381个推导氨基酸残基。序列分析表明，它与日本对虾（也称为日本囊对虾）的一种核酸酶在核酸水平上有83%的同一性，在氨基酸水平上有89%的同一性。对序列、保守基序和系统发育树的比较分析表明，斑节对虾核酸酶（PMN）属于DNA/RNA非特异性内切核酸酶（DRNSN）家族。使用针对PMN mRNA的特异性引物对七种不同对虾组织进行RT-PCR分析，结果显示正常对虾中该基因的表达仅限于肝胰腺。用正常对虾和感染白斑综合征病毒（WSSV）的对虾的肝胰腺mRNA对PMN进行半定量RT-PCR分析表明，在病毒感染早期，肝胰腺中PMN显著上调（P<0.05），但随着疾病明显症状的出现，其表达又恢复到基线水平。同时，该基因的表达始终局限于肝胰腺，在其他组织中从未检测到，包括那些据报道是WSSV主要靶标且感染后凋亡水平升高的组织。结果表明，PMN可能是一种消化酶，不太可能参与与凋亡相关的标志性DNA消化过程。
