The Study On The NOS And Related Factors Of Shrimp Litopenaeus Vannamei

Ambient temperature and disease were important factors which affected the healthy cultivation of Litopenaeus vannamei.L.vannamei is very sensitive to environmental changes and the outbreak of the diseases are also associated with temperature changes,so further study of its metabolic regulation mechanism and immune response to environmental stress and pathogens stimulation has great significance in the healthy aquaculture of L.vannamei.In the present study,corresponding biochemical methods were used to investigate the impact of temperature stress on the oxygen and energy metabolism in hepotopancreas of L.vannamei.The immune function of hemocyte and hepatopancreas was preliminary studied by RNA interference?RNAi?technology.The full length cDNA sequence of guanylyl cyclases?GC?which played an important role in the downstream of NOS regulation was cloned from L.vannamei and its molecular structures was analyzed.The tissue expression and temporal expression in hepatopancreas,nerve and hemocyte after injected with White Spot Syndrome Virus?WSSV?were investigated by Semi-quantitative PCR technology.The nitric oxide synthase?NOS?activity,nitric oxide?NO?and cyclic guanosinc monophosphate?cGMP?concentration in the hepatopancreas were investigated after the stimulation with WSSV.We also achieved high purity of NOS gene NO synthesis domain protein with His label by E.coli prokaryotic expression system based on the full length cDNA sequence of NOS in previous studies.Successful build a T7 phage display library which included ten kinds of different organizations without stimulation from L.vannamei.The results are as follows:?1?The impact of low and high temperature on the superoxide anion(O₂^-.)production,superoxide dismutase?SOD?activity,catalase?CAT?activity,glutathione?GSH?concentration,nitric oxide synthase?NOS?activity,nitric oxide?NO?and adenosine triphosphate?ATP?oncentration were examined.The results showed that O₂^-.production and corresponding antioxide enzymes could be induced significantly after cold and heat stress,but different antioxide enzymes might play different roles in shrimp response to thermal stress.The NOS activity and NO concentration increased significantly after heat stress,NO might play an important role in induction of many signaling pathways to response to thermal stress.In addition,more ATP was produced after cold and heat stress.Our findings indicated that thermal stress lead to oxygen metabolism disorder,which might be due to the stressed temperature beyond the oxygen-and capacity-limited thermal tolerance?OCLTT?capbility of L.vannamei.The disorder of oxygen metabolism might have an important impact on energy metabolism and other physiological activities of L.vannamei.?2?We synthesized sequence-specific double–stranded NOS which target sequence is the NOS gene NO synthesis domain of L.vannamei in vitro.The influence of dsNOS and the dual function of dsNOS and WSSV in the larvae of L.vannamei were detected after injection.The total hemocyte counts,NO concentration in hemocyte and hepotopancreas,cGMP concentration in hepatopancreas were investigated after injected with dsRNA and WSSV.The results showed that NOS expression was inhibited,the NOS activity and NO concentration in hepatopancreas decreased while the cGMP concentration was up-regulated after injected with dsNOS.?3?The full length cDNA of LvGCIII was 1641 bp,contains a 645 bp 5'untranslated region?UTR?,a 152 bp 3'UTR and an 855 bp open reading frame?ORF?encoding a predicted protein of 284 amino acids.The protein exhibits a Coiled coil and CYCc domain.The LvGCIII transcript appeared to be strongly expressed in intestine,never,brain,heart,skin and hepatopancreas,while the lower expression was found in stomach,muscle,hemocyte and gill.After injected with WSSV,the LvGCIII expression levels showed significant up-regulation in hepatopancreas?P<0.05?,but the change of expression levels was not significant in never and hemocyte.The NOS activity first up-regulated and then down-regulated at the end in the hepatopancreas,the NO and cGMP concentration showed a similar changing profile with NOS,but the cGMP concentration changed more significant compared with control?P<0.05?.?4?The recombinant plasmid pET-32a?+?-NOS was built,which contains NOS gene NO synthesis domain sequence of L.vannamei and His tag.We achieved and purified the fusion protein in vitro,got high purity of soluble pET-32a?+?-NOS recombinant protein.The fusion protein was identificated as NOS gene NO synthesis domain protein of L.vannamei by Western blot.?5?Selected hepatopancreas,muscle,never,intestine,stomach,heart,gill,eye stalk,hemocyte and skin tissues from healthy L.vannamei without stimulation.The extracted total RNA blend were used for the separation of mRNA.T7 phage display library successfully constructed with 87%insert rate and 70%fragment more than 250 bp.The library capacity reached 2.8x10¹⁶ pfu/mL after amplification.