Peepim Termsri, Phiwsaiya Kornsunee, Charoensapsri Walaiporn, Khunrae Pongsak, Senapin Saengchan, Rattanarojpong Triwit
Department of Microbiology, Faculty of Science, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand.
J Biotechnol. 2016 Feb 10;219:48-56. doi: 10.1016/j.jbiotec.2015.12.019. Epub 2015 Dec 19.
HtrA2 is an apoptosis-activating gene that enhances the apoptotic process by preventing the formation of the IAP-caspase complex, thereby freeing caspase to trigger the apoptosis pathway. In this study, we presented the full-length cDNA sequence of HtrA2 from Litopenaeus vannamei (LvHtrA2). The full-length LvHtrA2 was 1335 bp, encoding 444 amino acids. This deduced amino acid sequence contained five conserved domains: a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a trimerization motif, a serine protease domain, and a PDZ domain normally found in the HtrA2 proteins of other organisms. A phylogenetic analysis revealed that LvHtrA2 clustered with the HtrA2 from other invertebrates and was closely related to Penaeus monodon HtrA2 (PmHtrA2). RT-PCR with RNA extracts from L. vannamei revealed that LvHtrA2 expression was found in several tissues, including the lymphoid organs, the haemocytes, the hepatopancreas, the gill, and the stomach, with different expression levels. When determining the role of LvHtrA2 in WSSV infection, it was found that LvHtrA2 transcription was early up-regulated in the WSSV-infected shrimp at 8h post-infection (p.i.) and expression still remained high at 48 h p.i.. It also demonstrated that dsRNA specific to LvHtrA2 reduced the cumulative mortality in the WSSV-infected shrimp compared with the control group. Additionally, depletion of the LvHtrA2 transcripts reduced expression levels for caspase-3 (Cap-3) gene in shrimp. This result could suggest that LvHtrA2 may involved in apoptosis mediated mortality rather than providing immune protection during WSSV infection.
HtrA2是一种凋亡激活基因，它通过阻止IAP-半胱天冬酶复合物的形成来增强凋亡过程，从而使半胱天冬酶得以释放以触发凋亡途径。在本研究中，我们展示了凡纳滨对虾（Litopenaeus vannamei）的HtrA2（LvHtrA2）的全长cDNA序列。LvHtrA2全长1335 bp，编码444个氨基酸。该推导的氨基酸序列包含五个保守结构域：一个线粒体靶向信号（MTS）、一个跨膜（TM）结构域、一个IAP结合基序（IBM）、一个三聚化基序、一个丝氨酸蛋白酶结构域以及一个通常在其他生物体的HtrA2蛋白中发现的PDZ结构域。系统发育分析表明，LvHtrA2与其他无脊椎动物的HtrA2聚类，并且与斑节对虾HtrA2（PmHtrA2）密切相关。用凡纳滨对虾的RNA提取物进行RT-PCR分析表明，LvHtrA2在包括淋巴器官、血细胞、肝胰腺、鳃和胃在内的多个组织中均有表达，且表达水平各异。在确定LvHtrA2在白斑综合征病毒（WSSV）感染中的作用时发现，在感染WSSV的对虾中，LvHtrA2转录在感染后8小时（p.i.）早期上调，并且在感染后48小时p.i.时表达仍保持高水平。这也表明，与对照组相比，针对LvHtrA2的双链RNA（dsRNA）降低了感染WSSV的对虾的累积死亡率。此外，LvHtrA2转录本的缺失降低了对虾中半胱天冬酶-3（Cap-3）基因的表达水平。该结果可能表明，LvHtrA2可能参与凋亡介导的死亡，而不是在WSSV感染期间提供免疫保护。
