Functional Study Of Immune Related Genes QM And SERPIN From The Pacific White Shrimp, Litopenaeus Vannamei

Due to its fast growth, delicious taste, high disease resistance and other advantages, thePacific white shrimp, Litopenaeus vannamei was cultured in a large area in our country.However, with the expansion of the farming scale, disease problem is prominent day by dayand cause serious economic loss. Lacking of adaptive immunity compel shrimp to defenseagainst the invasion of pathogens only depending on innate immune system. Therefore, toexplore the disease resistance genes from the perspective of the innate immunity will laygroundwork for disease-resistant breeding in the future.Phenoloxidase activated system, consisting of phenol oxidase, protease, patternrecognition and protein inhibitor is an important recognition and defense system of thecrustaceans. The proPO-AS system recognized the non-self signal directly and throughinhibiting the extracellular protease and chitinase activity of pathogens strengthen hemocytephagocytosis, form nodular and cystn, mediated agglutination and condensation, producebactericidal substances to play an important role in wound healing, suppress and even kill ofpathogenic microorganisms. Therefore, clarifying the function of key molecular inphenoloxidase activated system has the vital significance for shrimp immunity research.In this study, gene specific primers were designed according to the homologous speciessequences and combining the technology of real-time fluorescent quantitative PCR,double-stranded RNA interference, prokaryotic expression vector construction, fusion proteinpurification and bioinformatics analysis, we obtained two genes LvQM and Lvserpin,analyzed their expression profile after pathogenic stimulation and explored their function inthe immune response, finally obtained the following results:(1) The cDNA sequence of LvQM is733bp with the open reading frame of663bpencoding220amino acids. Lvserpin cDNA sequence is1284bp with the open reading frameof1248bp encoding415amino acids.(2) The expression of LvQM was detected in all tested tissues and most highly expressedin gill, moderately expressed in ovary and muscle, and less expressed in hemocytes, eyestalk, hepatopancreas and intestine. The Lvserpin transcripts were most highly expressed in gill,moderately expressed in hemocytes, heart, intestine, stomach, testis, muscle, eyestalk, andlesser expressed in nerve and hepatopancreas.(3) In hepatopancreas and hemocytes, the LvQM transcripts were significantlyup-regulated in the early stages post infection of Vibrio anguillarum. In Micrococcuslysoleikticus challenged group, the expression of Lvserpin mRNA increased markedly at2hpost infection (hpi), then declined significantly to the bottom at6hpi, followed by an increaseand reached a second peak at48hpi. For V. anguillarum challenge, the expression of Lvserpinwas significantly increased and reached a peak at6hpi. Then, the expression level wasdecreased at12hpi and dropped back to the original level at24hpi. In the WSSV challengedgroup, the expression of Lvserpin was fluctuated, but no significant change was found before48h. At48hpi, the expression level was up-regulated and reached the highest in the wholeexperimental stage of WSSV challenge.(4) Knocked down LvQM mRNA expression in vivo by dsRNA-mediated RNAinterference, the expression of LvproPO was dramatically decreased comparing with thecontrol shrimps, and also the PO activity in shrimp hemolymph was significantly decreased,moreover, suppression of LvQM led to increased mortality. Silencing of Lvserpin significantlyincreased the transcription of PPAE, PPAF, proPO, ALF, Crustin and Pens3and also led toincreased mortality.(5) Injection of rLvQM significantly increased the PO activity in shrimp hemolymph andalso caused the enhancement of the bacterial clearance in shrimp. The rLvserpin could inhibittrypsin activity and the proteinase activity of trypsin could even be more completely inhibitedwith increasing concentration of rLvserpin. Also, rLvserpin could inhibit the growth of V.anguillarum with a dose-dependent manner. Furthermore, the rLvserpin might have animportant inhibitory role in the shrimp prophenoloxidase system.The above results implied that LvQM was involved in the shrimp immune defensethrough the activation of phenol oxidase system with the regulation of PO activity. AndLvserpin was likely to have capability in protecting shrimps from pathogen invasion throughinhibiting bacterial proteinases, modulating antimicrobial peptide production or regulating theproteinases involved in proPO activation.