Molecular characterization and function analysis of a nucleotide excision repair gene Rad23 from Litopenaeus vannamei after Vibrio alginolyticus challenge.

Nucleotide excision repair (NER) removes many different types of DNA lesions, and NER related host factors are reported to aid recovery steps during viral integration. Here, we report the identification and characterization of a DNA repair gene Rad23 from Litopenaeus vannamei and explore its role in innate immunity of crustaceans. LvRad23 contains a1149 bp open reading frame (ORF) which encodes a 382 amino acids protein with predicted theoretical isoelectric point of 4.21. LvRad23 was ubiquitously expressed in the muscle, eyestalk, gill, stomach, heart, legs, intestine, and hepatopancreas in order from high to low and LvRad23 protein was showed to be located in the cytoplasm of Drosophila S2 cells. The homology analysis showed that it has a high sequence homology with Rad23 protein from Marsupenaeus japonicus. Vibrio alginolyticus challenge induced a remarkable up-regulation of LvRad23 mRNA in hepatopancreas. Knocking down LvRad23can interfere the NER pathway by down regulating the expression of replication protein A (RPA) and proliferating cell nuclear antigen (PCNA). However it didn't cause any significant difference on total hemocyte count (THC) between LvRad23-silenced and non-silenced group.LvRad23-silenced then challenge with V. alginolyticus inducing high level of reactive oxygen species (ROS) and DNA damage in hemolymph. As well as decreased THC, which seriously diminished the innate immune system of L. vannamei. Meanwhile, the NER pathway was reactived by enhancing the expression of LvRad23 and promoting the production of LvPCNA to resist apoptosis and maintain proliferation of hemolymph cells in the later stage. Our results suggest that LvRad23 plays a vital role in shrimp specific immune response to V. alginolytcus through its participation in NER pathway.

Effect of tributyltin chloride (TBT-Cl) exposure on expression of HSP90ß1 in the river pufferfish (Takifugu obscurus): Evidences for its immunologic function involving in exploring process.

HSP90ß1 (known as glyco-protein 96, GP96) is a vital endoplasmic reticulum (ER) depended chaperonin among the HSPs (heat shock proteins) family. Furthermore, it always processes and presents antigen of the tumor and keeps balance for the intracellular environment. In the present study, we explored the effect of tributyltin chloride (TBT-Cl) exposure on HSP90ß1 expression in river pufferfish, Takifugu obscurus. The full length of To-HSP90ß1 was gained with 2775 bp in length, with an ORF (open reading frame) encoding an 803 aa polypeptide. A phylogenetic tree was constructed and showed the close relationship to other fish species. The HSP90ß1 mRNA transcript was expressed in all tissues investigated with higher level in the gill and liver. After the acute and chronic exposure of TBT-Cl, the To-HSP90ß1 mRNA transcript significantly was up-regulated in gills. Moreover, the histology study indicated the different injury degree of TBT-Cl in liver and gill. Immunohistochemistry (IHC) staining results implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-HSP90ß1 to TBT-Cl exposure. All the results indicated that HSP90ß1 may be involved in the resistance to the invasion of TBT-Cl for keeping autoimmune homeostasis.