Characterization And Function Analysis Of Integrin β,Rh Protein,Na~+/H~+-exchanger Related To Ammonia Nitrogen Tolerance In Penaeus Monodon

In order to study the molecular mechanism of ammonia nitrogen resistance of Penaeus monodon,the full length of cDNA of three related genes in ammonia nitrogen stress pathway and ammonia nitrogen detoxification metabolic pathway of Penaeus monodon were cloned for the first time by RACE technique,and bioinformatics analysis was carried out.The expression patterns of three genes in various tissues and under 96h acute ammonia nitrogen stress were analyzed by QPCR technique,which proved that there was a correlation between the three genes and ammonia nitrogen stress.PmItgb is a differentially expressed gene screened from the ammonia nitrogen stress transcriptome library of Penaeus monodon constructed in our laboratory.Some studies have shown that Itgb is a multi-functional protein involved in the process of growth and development,immunity and apoptosis.In this paper,the effects of PmItgb on the growth and development,molting stages,innate immunity and ammonia nitrogen-induced apoptosis of Penaeus monodon were studied.To some extent,our results show that PmItgb gene is associated with the slow growth and development and the decline of innate immune system function caused by ammonia nitrogen stress.Pm Rh and Pm NHE genes were selected in the ammonia nitrogen detoxification metabolic pathway,and RNAi technique was used to explore the relationship between them and other related genes related to ammonia transport and excretion,as well as the relationship between silencing of these two genes and death rate in high ammonia nitrogen environment.It laid a foundation for studying the role of Pm Rh gene and Pm NHE gene in the detoxification metabolism of Penaeus monodon.The 3011 bp cDNA sequence of PmItgb was cloned from P.monodon using rapid amplification of cDNA ends（RACE）PCR.Phylogenetic tree analyses indicated that the amino acid sequence of PmItgb should be merged into Fenneropenaeus chinensis（93%similarity）.Quantitative real-time PCR revealed that PmItgb mRNA was highly expressed in the hemolymph.In addition,with regard to developmental stages,PmItgb showed significantly higher expression in oosperm,nauplius IV,zoea I and III,and postlarval stages than that in other development stages.PmItgb expression in the shrimp epidermis was higher in the postmolt（B）stage,and lower in other molting stages.We also found that Vibrio harveyi and V.anguillarum challenge enhanced PmItgb expression in the hepatopancreas and gills.When PmItgb was inhibited,innate immunity-related genes such as ALF,crustin 1,crustin 7,Penaeidin 3,and Penaeidin5 were significantly down-regulated.Furthermore,we demonstrated that PmItgb knock-down by specific ds RNA reduced bacterial clearance.In high ammonia nitrogen concentrations,PmItgb was significantly up-regulated in the hepatopancreas and gills.After PmItgb was silenced,the rate of mortality owing to high ammonia nitrogen concentrations decreased;the expression of related anti-apoptotic genes was up-regulated,and that of the apoptotic genes was slightly down-regulated.These results suggested that PmItgb may be involved in shrimp innate immunity and mediate apoptosis of hepatopancreatic cells induced by high ammonia nitrogen environments.The full-length cDNA of Rh protein（Pm Rh）gene of Penaeus monodon is 1946 bp（Gen Bank number:MT164533）,including the open reading frame ORF 1476bp,5’non-coding region 264 bp,3’non-coding region 206bp.It encodes 491 amino acids with a molecular weight of 160.57KDa and a theoretical isoelectric point of 4.85.The sequence contains 13 phosphorylation sites,3 glycosylation sites and 12transmembrane domains.The sequence contains an ammonium transport functional domain located at 60-462 aa.Phylogenetic tree analysis showed that Pm Rh was most closely related to the Rh type A like protein that of Litopenaeus vannamei,and the highest similarity was 92.26%.Q RT-PCR showed that the expression of Pm Rh mRNA was the highest in Gill,and the expression was specific.At high concentration of ammonia and nitrogen,the expression of Pm Rh in Gill,hemolymph and muscle was significantly up-regulated.After Pm Rh silencing,the blood ammonia concentration and mortality increased caused by high concentration of ammonia nitrogen.The inhibition of Pm Rh could down-regulate the mRNA expression pattern of NKA,K⁺-channel,AQP,CA and VAMP,which had the function of ammonia-nitrogen transport under high ammonia-nitrogen stress,while the Pm NHE gene was significantly up-regulated,and the expression of GS gene,which was closely related to ammonia-nitrogen detoxification metabolism,was significantly up-regulated.It can be inferred that Pm Rh may be at the core of the metabolic regulation mechanism of ammonia nitrogen in Penaeus monodon.Ammonia-nitrogen metabolism involves complex interactions between a variety of proteins,which can transport two different molecular forms of ammonia NH₃ and NH₄⁺,Pm Rh proteins.The complex interactions between NH₃ and NH₄⁺proteins and other proteins enable the coordination of NH₃ and NH₄⁺transport,and contribute to the fine regulation of ammonia nitrogen transport and excretion in the gills of Penaeus monodon.The full-length cDNA of the sodium-hydrogen ion transporter（Pm NHE）gene of Penaeus monodon is 2788 bp（Gen Bank number MT164534）,including the open reading frame ORF 2643bp,the 5’non-coding region 76,the bp,3’non-coding region78 bp,which encodes 877 amino acids,its molecular weight is 97.97 KDa,and the theoretical isoelectric point is 6.54.There are 23 phosphorylation sites,6 glycosylation sites and 12 transmembrane domains.The sequence contains a sodium/proton exchanger domain located at 78-490 aa.Phylogenetic tree analysis showed that the phylogenetic relationship between Penaeus monodon and Litopenaeus vannamei was the most close and clustered into one branch,and the highest similarity was 93.92%.Quantitative analysis of various tissues showed that Pm NHE was expressed in a variety of tissues,the expression of Pm NHE was the most abundant in the intestine,and the expression in muscle was only second to that in the intestine.The quantitative results showed that under high concentration of ammonia nitrogen stress,the expression of Pm NHE in Gill tissue of Penaeus monodon was significantly up-regulated and Pm NHE in intestinal tissue was significantly down-regulated.The silence of Pm NHE could increase the death rate of Penaeus monodon in high ammonia nitrogen environment.These results provide a basis for further study on the role of Pm NHE in Penaeus monodon under ammonia nitrogen stress.