Study On Physiological, Biochemical And Molecular Mechanism Of Enchanced Immunity Ability By Chitosan In Ayu(plecoglossus Altivelis)

Plecoglossus altivelis is an annual spicy of migratory fish and it has been widely cultured fish with theimprovement of breeding and economic value. The growth of Plecoglossus altivelis is restricted bytemperature of water and light intensity. With the agriculture scale constantly expanding，the water foragriculture has become more polluted, which leads to frequent fish disease. The healthy development ofagriculture faces various stresses. Therefore, it remains primary importance in the investigation ofimmunogenes and immunopotentiator to get out current dilemma.Molecular chaperones-heat shock protein70kDa （HSP70s）, one of the most important proteins amongmembers of heat shock proteins, could be induced by stimulus of environment, physiology andpathophysiology. HSP70s play important roles in folding, disaggregation and translocalization of neonateprotein, and they also participate in cellular growth, differentiation and apoptosis. More strikingly, they canprotect the cell, organ, even organism from destructive stimulus motivated by heat, hypoxia and bacterialinfections.In this thesis, the main emphasis was the effects of chitosan on the immune function of organ.The hsp70was cloned from PA using RACE technique and analyzed by bioinformatic softwares. The realtime RT-PCR was performed to study the chitosan affection on the expression of immune genes,approaching the mechanism of physiology, biochemistry and molecule.The aims of this study were to investigate immunostimulants impacts on the growth and nonspecificimmunity of Plecoglossus altivelis with feeds mixed chitosan, through the assessment of the activities ofpreoxidase （POD）, lysozyme （LSZ）, acid phosphatese （ACP）, and statistical analysis of red cells and whitecells in blood. Firstly, the highest number of red cells had been observed in Plecoglossus altivelis exposedto0.5%chitosan, but the number in other groups showed a trend of decline. The maximum amount ofwhite cells appeared at0.5%chitaosan. Furthermore, the activity of LSZ in muscle had no evident change,but was significantly enhanced in the serum, spleen, liver, kidney and gill. The activity of LSZ increased tothe highest at1%chotisan. Lastly, there were significant differences of POD and ACP activities in muscle,serum, spleen, liver, kidney and gill. The peak activities of POD and ACP appeared at1%chotisan.Considering the remarkably effects on improving the immunity, the optimized concentration of chotisan is1%.The complete cDNA was obtaind by RACE techniques from healthy Plecoglossus altivelis. The fulllength of hsp70in Plecoglossus altivelis was2361bp. According to analysis of bioinformatics, there wasan open reading fragment of1956bp sequence encoding a polypeptide of651amino acids with theestimated molecular weight of71.22kDa and isoelectric point （PI） of5.16. The amino acids sequence ofPlecoglossus altivelis showed94%identity with Salmo salar, Lutjanus sanguineus, Paralichthys olivaceus, Megalobrama amblycephala, Hypophthalmichthys molitrix, Ctenopharyngodon idella and Danio rerio. TheHSP70contained five functional motifs: repeats of tetrapeptide GGMP and typical HSP70signaturemotifs, ATPase binding site, Ca²⁺binding site, and there was a motif of cytoplasmic localization with"EEVD" at the C-terminus. The predicted secondary structure composition for the protein had about41.63%alpha helixes,27.65%extended strand,13.67%beta turn and33.03%random coil. The tertiarystructure of HSP70was performed by SWISS-MODEL with homology models, and indicated thatthree-dimensional structure of EF was similar to typical tertiary structure of enzyme, with a fracturestretching across two leaflike structures.The deduced amino acid sequence was highly similar to theHSP70s of other fishes. HSP70s in fishes were located on the backbone of evolution branch in the tree, andless related to HSP70s from mammals which were in a different branch. HSP70and Oncorhynchus mykissformed a cluster. The pylogenetic tree suggested that PaHSP70and other HSP70s might have a commonancestor in the evolution.Semi RT-PCT was employed to examine gene expression in various tissues of Plecoglossus altivelis.Pahsp70s were distributed in gill, intestinum, liver, kideney spleen and muscle, but expressed highly inliver, kidney and spleen, which suggested that Pahsp70s might be associated with immune function.Under the treatments of different concentration of chitosan, mRNA expressions of three kind ofimmunity genes were quantified by real-time quantitative PCR （q RT-PCR）. When the Plecoglossusaltivelis were kept in lower concentration of chitosan, the relative expressions of immunogenes （hsp70,lect2and nkef） in liver were increased slightly. The expression level was highest in1%chitosan, and thendecreased gradually as the concentration increased. The change tendency of lect2in kidney and spleen wasaccordant with that in liver. The expression level of lect2increased at0.5%chitosan, and reached themaximum at1%chitosan, but then declined at higher concentration. The change of expression for nkef inliver was significantly different, and the peak of expression appeared at1%chitosan. But in spleen, theeffect of chitosan on the relative expression of nkef was insignificant.The highest expressions of three immune genes were found at the concentration point of1%, which wasconsistent with the maximal immune indexes including LZS, POD and ACP at1%chitosan. The analysisled to the following conclusion: chitosan might have immune-enhancing properties on Plecoglossusaltivelis, via regulating the expression of immune genes and increasing the immune activity of organ.