Functional Identification Of Chitinase Gene Family And Its Immune Mechanism In Response To Salinity Changes In Portunus Trituberculatus

Portunus trituberculatus is an important marine economic crab,and White Spot Syndrome Virus(WSSV)and Vibrio parahaemolyticus were the main pathogens causing its death.Salinity is an important environmental factor in the cultivation of P.trituberculatus.As a result of rainy weather and large water change,the salinity of the breeding water often changes sharply,resulting in the stress reaction of P.trituberculatus,physiological metabolism disorder,and then the decline of immunity,easily causing the outbreak of diseases and even a large number of deaths.Chitinase is a kind of key enzyme widely existing in arthropods,which is involved in physiological processes such as ecdysis growth,abnormal development and immune response.Chitin can be cleaved to play a variety of functions.Chitinases in arthropods belong to the polygenic family of 18-glucosidase hydrolase(GH18),most of the current studies focus on insects,and its members are classified into eight groups(1-8).Compared with insects,crustaceans are relatively lagging in this field.Only seven groups of chitinase genes have been found so far.The study on chitinase gene of P.trituberculatus has just started,with only a few cases such as PtCht3(Group 3),PtCht(Group 7)and PtChti(Group 3).The functional studies mainly focus on molting and salinity adaptation.On this basis,this study cloned five P.trituberculatus chitinase gene family,categorize and analyze its structure features,using quantitative real-time PCR(RT-qPCR)realize the preliminary identification of the gene function,using fluorescent in situ hybridization(FISH),prokaryotic expression and bacteriostatic experiment to verify gene immune function.To explore the immune mechanism of genes in response to changes in salinity by injecting different pathogens,the main research contents are as follows:1.Cloning and sequence analysis of five chitinase genes of P.trituberculatusIn this study,RACE(Rapid-amplification of cDNA ends)was used to clone five chitinase genes,named as PtCht-1,PtCht-2,PtCht4,PtCht6,and PtCht8,respectively.Among them,the total length of PtCht-1 cDNA was 1910 bp and ORF 1749 bp,encoding 582 amino acids.The predicted molecular weight(MW)was 65.122 kDa,and the theoretical isoelectric point(pI)was 4.52.The total length of PtCht-2 cDNA was 2422 bp and ORF1851 bp,encoding 616 amino acids,and predicting MW 69.122 kDa and pI 4.62.The total length of PtCht4 cDNA was 1549 bp and ORF 1389 bp,encoding 462 amino acids,and predicting MW 51.172 kDa and pI 5.03.The total length of PtCht6 cDNA was 2736 bp,ORF 2103 bp,encoding a total of 700 amino acids,and predicting MW 76.603 kDa,pI 6.29.The total length of PtCht8 cDNA was 1973 bp,ORF 1599 bp,and a total of 532 amino acids were encoded.It was predicted that MW 59.867 kDa and pI 6.79.P.trituberculatus five chitinase gene encoding amino acid sequence respectively with other species of chitinase gene encoding protein homology comparison analysis,the results showed that PtCht-1 amino acid sequence has the highest homology with EsCht4(Eriocheir sinensis),65%;PtCht-2,PtCht4,and PtCht6 amino acid sequences have the highest homology with SpCht4(Saylla paramamosain),96%,100% and 85%,respectively;PtCht8 has the highest homology with LmCht5(Locusta migratoria),98%.Phylogenetic analysis of the amino acid sequences of P.trituberculatus was carried out with MEGA 6.0 software.The results showed that chitinase genes of crustacean were divided into seven groups,PtCht-1 and PtCht-2 belonged to Group4,PtCht4,PtCht6 and PtCht8 belonged to Group6,Group5 and Group1,respectively.This study further improved the structure of chitinase GH18 gene family in P.trituberculatus,and provided reference materials for further study on chitinase polygene family in crustacean.2.Preliminary identification of five chitinase genes in P.trituberculatusAccording to research,the main functions of chitinase in crustaceans are mainly reflected in the participation in molting,the digestion of chitinfood in the intestinal periphage membrane and the regulation of pathogen immunity.The expression patterns of chitinase genes PtCht-1,PtCht-2,PtCht4,PtCht6 and PtCht8 in nine tissues(heart,hepatopancreas,blood cells,gill,stomach,intestine,muscle,eyestalk and cuticle)were analyzed by RTqPCR,and the gene function was preliminarily explored.The results showed that five genes were all expressed in nine tissues,among which PtCht-1,PtCht-2 and PtCht6 were the most highly expressed in hepatopancreas,PtCht4 was in intestines,and PtCht8 was in epidermis.Therefore,it is speculated that PtCht-1,PtCht-2 and PtCht6 genes are most likely to participate in the pathogen defense of the body as immune genes and play a role in immune function.In addition to the function of digestion and absorption of chitin in chitin-food,PtCht4 gene may also participate in the immune defense of the body.PtCht8 gene may be mainly involved in the molting development of P.trituberculatus.This result has laid a theoretical foundation for the further study of chitinase gene function in P.trituberculatus.3.Immune function verification of chitinase gene in P.trituberculatusThis study through the gene nucleotide sequence design synthesis PtCht-1,PtCht-2 and PtCht6 gene FISH probe,will inject WSSV and V.parahaemolyticus infected P.trituberculatus hepatopancreas tissue paraffin embedding slice,processing after staining fluorescence microscope photograph,the results showed that three genes in P.trituberculatus hepatopancreas cells are distributed in cytoplasm and nuclei,and gene location near the lumen after infection pathogens shows obvious aggregation,and in the same direction with hepatopancreas playing transshipment bubble movement during the immune function,suggests PtCht-1,PtCht-2 and PtCht6 genes may act as immune genes to participate in the pathogen defense mechanism of P.trituberculatus.In addition,by constructing the PtCht4 gene of prokaryotic expression vector PtCht4-pET28 a,thus induced protein expression,purification and renaturation,using different concentrations after renaturation of protein PtCht4 bacteriostatic experiment,the results show that with higher protein concentration,bacteriostatic rate increased significantly,and the same concentration of protein in pair of V.parahaemolyticus rate was significantly higher than that of Staphylococcus aureus,suggests PtCht4 has obvious inhibiting effect proliferation of pathogenic bacteria,verified the genes as immune genes involved in the body's immune defenses.The results of this study will lay a foundation for the subsequent in-depth study on the mechanism of the immune genes of P.trituberculatus to play the role of immune function.4.Study on the effect of chitinase gene on immunity of P.trituberculatusSalinity is an important environmental factor in the breeding of P.trituberculatus.In order to study whether PtCht-1,PtCht-2,PtCht4 and PtCht6 genes play a certain role in the effect of low salinity on the immunity of P.trituberculatus,we set up the experiment of pathogen infection under natural conditions and low salinity stress.It was found that under low salinity stress,the expression of PtCht-1,PtCht-2 and PtCht6 genes mainly were significantly down-regulated in the hepatopancreas and blood before 48h(P<0.05),however,the expression of PtCht4 gene was significantly downregulated in the hepatopancreas before 72h(P<0.05),with a maximum down-regulation of 73 times.After WSSV injection under low salinity stress,the expression of PtCht-1 in the hepatopancreas and blood cell both reached the peak at 72 h,and the expression of PtCht-2,PtCht4 and PtCht6 reached the peak at 24 h and 72 h,respectively,PtCht-1 was significantly later than the peak time of hepatopancreas infected WSSV under normal conditions(the maximum delay was 24h),PtCht-2 and PtCht4 was significantly later than the peak time of hepatopancreas infected WSSV under normal conditions(the maximum delay was 69h),however,PtCht6 was significantly later than the peak time infected WSSV under normal conditions(the maximum delay was 12h).After injected with V.parahaemolyticus under low salinity stress,the mortality rate per unit time was significantly increased(P<0.05)compared with that of V.parahaemolyticus infected with the same concentration of V.parahaemolyticus under normal conditions(all died after 72h)due to its strong lethality to P.trituberculatus(P<0.05).The results indicate that low salinity stress may inhibit or delay the normal expression of chitinase and other immune genes,thus leading to the decline of immunity.The results of this experiment provide more reference data for further exploring the immune function of chitinase in crustaceans.