| In the world aquaculture industry,P.vannamei is the main culture shrimps.With the rapid development of culture scale and the gradual deterioration of culture environment,the outbreak of vibrio disease in water has become the main factor limiting shrimp culture.Based on the transcriptome analysis,it was found that the expression of peritrophic membrane-related genes in P.vannamei changed significantly after being challenged by Vibrio parahaemolyticus.The peritrophic membrane is an important physical barrier secreted by invertebrate midgut cells.It not only separates the intestinal lumen and midgut cells in structure,but also can protect the midgut epithelial cells and prevent the invasion of harmful substances.It also has the functions of fixing digestive enzymes,intestinal regionalization,toxin binding and antioxidation,and allows digestive enzymes to enter the intestinal cavity freely to promote nutrient absorption.In order to further study the function of peritrophic membrane-related genes in anti-V.parahaemolyticus to P.vannamei,the bioinformation and expression of peritrophic membrane family genes were analyzed.The main results are as follows:1.Analysis of sequence characterization of peritrophic membrane proteins of P.vannameiBased on transcriptome data of P.vannamei 25 peritrophic membrane related genes(brp 3,comp,chit-3,chit-1,cp-2,egg 2A,k-16,p-1A,p-1B,p-48,p-55,pro 10,PERK8,pro-E A,pro-E B,pro 4,Sgs-3,SP65,zon,cp 19,cp-AMP1 A,cp AM1199,cp AM1159,pro-re,ext)were identified.The sequence length of peritrophic membrane proteins of P.vannamei is between 103～1307 aa,therein,cp AM1159 and cp AM1199 are the shortest for only 103 aa and 125 aa,while brp3 and comp are the longest for 1307 aa and 1247 aa respectively.Their molecular weight is between 11.478～143.891 k Da.The isoelectric point ranges from 4.04 to 7.88,and most of the members are acidic proteins.Gene structure analysis showed that these genes had different numbers of exons and introns,therein,sp65 had the minimum exons with 2 exons,and some genes such as comp had the 23 exons and 22 introns.2.Analysis of conserved domains and motifs of peritrophic membrane proteinsof P.vannameiThe results of conservative domains and motifs analysis showed that there were19 genes with CBD2 domain(brp 3,comp,chit-3,chit-1,cp-2,egg 2A,k-16,p-1A,p-1B,p-48,p-55,pro 10,PERK8,pro-E A,pro-E B,pro 4,Sgs-3,SP65,zon),and 6genes with CBD4 domain(cp 19,AMP1A-like,cp AM1199,cp AM1159,pro-re,ext).Additionally,the results also found that the motif.1 was existed in these genes with CBD2 domain,while the motif.5 was shared in these genes with CBD4 domain,indicating that motif.1 and motif.5 may be involved in the formation of CBD2 and CBD4 type domains,respectively.3.Phylogenetic analysis of peritrophic membrane gene family of P.vannameiPhylogenetic tree analysis showed that most of the genes containing CBD2 domain were in different evolutionary branches from CBD4 domain,and their clustering branches were more reliable,while chit-3 and chit-1 may have mutated in the process of evolution and were far away from other CBD2 domain genes.4.Expression of peritrophic membrane genes in different tissues of P.vannameiThe results of semi-quantitative PCR showed that in healthy tissues,the expression of perineal membrane-related genes was the highest in stomach and intestine,followed by gill and hepatopancreas,and lower in heart,blood,muscle,brain and eye stalk.After being challenged by V.parahaemolyticus,it was found based on q RT-PCR atdifferent time points that there were 8 genes(comp,chit-3,chit-1,cp2,pro E-A,Sgs3,ext,zon,pro-E B)with increased expression in intestinal tract and 6 genes(pro E-A,pro4,cp2,pro E-A,pro4,p-1 A)in hepatopancreas;There were 4 genes(k 16,p55,pro 4,p-1 B)in the gut and 7(brp3,k 16,p-1 B,p-48,Sgs3,sp65,ext)in the hepatopancreas whose expression increased first and then returned to normal levels.These genes may be stimulated after challenge and up-regulated to participate in the immune response.There were 6 genes(brp3,sp65,p-1 A,cp19,cp AM1199)in intestinal tract and 1 gene(chit-3)in hepatopancreas whose expression decreased first and then increased.There was 1 gene(p-48)in the intestine and 2 genes(comp,zon)in the hepatopancreas that the expression of V.parahaemolyticus decreased significantly after challenge.That is,after the challenge of V.parahaemolyticus,in the intestine and hepatopancreas of P.vannamei,most of the peri-food membrane genes are up-regulated or down-regulated in varying degrees,indicating that after the body is invaded by pathogens,the peritrophic membrane genes of P.vannamei may play an important role in resistance to foreign pathogens and immune protection.5.Prokaryotic expression of peritrophic membrane genes pmur1 B and cp AM1159 of P.vannameiIn this study,the recombinant plasmids of p ET-32a-p-1B and p ET-32a-CPAM1159 were successfully constructed,and induced in E.coli expression strain BL21(DE3)for expression... |
