The Roles And Characterizations Of NOS And Related Immune Factors In Litopenaeus Vannamei Immune Response

In this paper, the change of some important immune factors in Pacific white shrimp,Litopenaeus vannamei were investigated after challenged by pathogen associatedmolecularpatterns (Laminarin, LPS, poly I:C), Vibrio parahaemolyticus and WSSV. thefull-length cDNA of Nitric Oxide Synthase (NOS), arginine kinase (AK) and two types ofcalmodulin (LvCaM-long and LvCaM-short) were cloned and the structures characterizationswere studied, the NO synthase domain of NOS, LvCaM-long and AK were expressed in E. coli,and the interaction protein with AK was also studied. The results are as follows:Immune challenge was performed by injecting three pathogen associated molecular patterns(Laminarin, LPS, poly I:C), V. parahaemolyticus and WSSV to pacific white shrimp to researchthe roles and mechanism of the PRRs, pro-phenoloxidase-activated system, antioxide system andanti-microorganism system in immune response. These results indicated that different immunefactors paly different roles in shrimp immune defense.A full-length cDNA of NOS (named as LvNOS) was obtained from Pacific white shrimp, L.vannamei, by RT-PCR and RACE-PCR. It was of4680bp, including a5’-teminal untranslatedregion (UTR) of278bp, a3’-terminal UTR of862bp and an open reading frame (ORF) of3540bp encoding a polypeptide of1179amino acids. It contained a typical NO synthase domain at theN-terminal, next to a flavodoxin1domain, a flavin adenine dinucleotide (FAD) binding domain,respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domainstructure at the C-terminal. Quantitative real-time reverse transcription PCR analysis revealedLvNOS to be expressed in most shrimp tissues, with highest expression in the hepatopancreasand weakest expression in skin. The changes of NOS expression and enzyme activity afterchallenge with V. parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas. Theresults indicated that the NOS expression and enzyme activity could be inhibited in hemocytesand hepotopacreas injection with V. parahaemolyticus and WSSV. These results suggested thatNOS might play an important role in shrimp’s defense against pathogenic infection. Then, theNO synthase domain gene was expressed in E. coli successfully, which made condition for futurestudy of NOS protein.The full-length AK cDNA was1446bp encoding356amino acids, and belonged to theconserved phosphagen kinase family which contained the ATP-gua PtansN domain and ATP-gua Ptrans. The whole ORF of AK was expressed in E. coli to get a protein having enzyme activity.The quantitative real-time reverse transcription PCR and Western-blot analysis revealed a broadexpression of AK transcripts and AK protein with the highest expression in the muscle and thelowest in the skin. The expression of AK transcripts and enzyme activity changed significantlyafter injection with V. parahaemolyticus and WSSV and the content of ATP decreasedremarkably. These results indicated that the AK played an important role in shrimp immuneresponse. It was found that the AK protein was interacted with shrimp actin T2revealed by GSTpull-down, mass spectrometry assays and two yeast hybrid system.Two types of CaM (named LvCaM-long and LvCaM-short) were cloned from L. vannamei.The full length of LvCaM-long gene was1101bp which had a ORF of450bp encoding149amino acids and LvCaM-short gene has a689bp cDNA which had a ORF of510bp encoding169amino acids respectively. Real-time PCR revealed that the LvCaM-long gene has a brodexpression in shrimp tissues, with highest in nerve, weakest in skin and the LvCaM-short genewas only founded in hepotopancres. After challenged by V. parahaemolyticus and WSSV, thetwo types of CaM expression changed significantly but differently, indicating that LvCaM-longand LvCaM-short played different roles in shrimp immune response. After that, LvCaM-longgene was expressed by E. coli for future study.