Abstract：
Litopenaeus vannamei Smad5 (LvSmad5) in cytoplasm has been proved to be involved in environmental stress response. As LvSmad5 could also locate in nucleus under specific stress, it was conjectured that LvSmad5 might participate in environmental stress response. While, the experimental evidence is still lacking. In this study, cytosolic LvSmad5 mutant or nuclear LvSmad5 mutant was expressed in Drosophila S2 cells, and then transcriptomic analysis of mentioned cells was performed using Illumina HiSeq based RNA-Seq, to reveal the function of LvSmad5 in nucleus. By comparing the two groups of cDNA libraries from S2 cells with cytosolic or nucleus LvSmad5 mutant, 86 differentially expressed genes as well as 765 differentially expressed transcripts were found. It was revealed that genes in the ER-stress response pathway, such as unfolded protein response and ER-associated degradation (ERAD) were enriched. Additionally, some kinds of metabolic reprogramming occurred in S2 cells with over-expressing nuclear LvSmad5, for significant changes in the expression of some metabolism-related genes. To test our infer that nuclear LvSmad5 was engaged in environmental stress response, homologous gene of Drosophila translocation in renal carcinoma on chromosome 8 in L.vannamei (LvTRC8) was chosen for further investigation. And studies about LvTRC8, a member of ERAD showed that it was induced by ER-stress or heat shock treatment. Suppressed the expression of LvTRC8 increased the cumulative mortality of shrimp upon stress. In some degree, these results support our speculation that nuclear LvSmad5 are involved in the environmental stress response of L. vannamei in fact.
摘要：
凡纳滨对虾Smad5(LvSmad5)在细胞质中的表达已被证明参与环境应激反应。由于LvSmad5也可以在特定应力下定位在细胞核中，推测LvSmad5可能参与环境应激反应。同时,实验证据仍然缺乏。在这项研究中,胞质LvSmad5突变体或核LvSmad5突变体在果蝇S2细胞中表达，然后使用基于IlluminaHiSeq的RNA-Seq对上述细胞进行转录组学分析，揭示LvSmad5在细胞核中的功能。通过比较具有胞浆或核LvSmad5突变体的S2细胞的两组cDNA文库，发现86个差异表达基因以及765个差异表达转录本。研究表明，ER-应激反应途径中的基因，如未折叠蛋白反应和ER相关降解(ERAD)被富集。此外,在核LvSmad5过表达的S2细胞中发生了某些代谢重编程，导致某些代谢相关基因的表达发生了显着变化。为了测试我们的推断，核LvSmad5参与了环境应激反应，选择凡纳滨对虾（LvTRC8）8号染色体上的肾癌中果蝇易位的同源基因进行进一步研究。关于ERAD成员LvTRC8的研究表明，它是由ER应激或热休克治疗引起的。抑制LvTRC8的表达增加了虾在胁迫下的累积死亡率。在某种程度上,这些结果支持了我们的推测，即核LvSmad5实际上参与了南美白对虾的环境应激反应。
