| Channel catfish(Ictalurus punctatus),also known as channel catfish introduced from North America to China,has become the main freshwater aquaculture species in China.While the number of breeding is rising,followed by the discomfort of the living environment,frequent diseases,production declined and other issues to some extent hindered the healthy breeding of channel catfish.It is particularly important to clarify the interaction mechanism between pathogens and hosts and the immune molecular mechanism of channel catfish,which will lay the foundation for seeking disease prevention and control methods.Diseases have always been the main obstacle to the healthy growth of fish.Yersinia ruckeri is one of the susceptible pathogens of channel catfish.This study will focus on transcriptome sequencing analysis of Y.ruckeri infection in channel catfish kidney in the process of interaction between the two reactions,in-depth understanding of the internal relationship between the two.In the analysis of transcriptome data,it was found that the immune-related JAK-STAT signaling pathway was significantly enriched at 6and 12 h in the KEGG enrichment analysis,indicating that immune-related genes were actively expressed,and interleukin was one of them.Previous studies had shown that most cytokines in animals and immune defense function complement each other,interleukin in the body involved in inflammation and other stimulate played an immune regulation,promote the maturation and differentiation of T lymphocytes and the role of body repair.Among them,IL-22,IL-10 and IL-6 are important cytokines in JAK-STAT signaling pathway,and there is no report on related studies in channel catfish.Therefore,this paper intends to study the interaction between I.punctatus and Y.ruckeri,to clarify the reaction after fish infection,and how pathogens cause fish disease.At the same time,the immune related genes IL-22,IL-10 and IL-6 in JAK-STAT signaling pathway were cloned and analyzed,and the gene expression levels under different stimulation were detected,so as to reveal the response dynamics of different genes after stimulation at the molecular level and further understand whether they participate in the immune response.1 Dual RNA-Seq of trunk kidneys extracted from channel catfish Infected with Yersinia ruckeri reveals novel insights into host-pathogen interactionsHost-pathogen intectarions are complex,involving large dynamic changes in gene expression through the process of infection.These interactions are essential for understanding anti-infective immunity as well as pathogenesis.In this study,the hostpathogen interaction was analyzed using a model of acute infection where channel catfish were infected with Y.ruckeri.The infected fish showed signs of body surface hyperemia as well as hyperemia and swelling in the trunk kidney.Double RNA sequencing was performed on trunk kidneys extracted from infected channel catfish and transcriptome data was compared with data from uninfected trunk kidneys.Results revealed that the host-pathogen interaction was dynamically regulated and that the hostpathogen transcriptome fluctuated during infection.More specifically,these data revealed that the expression levels of immune genes involved in Cytokine-cytokine receptor interactions,the NF-kappa B signaling pathway,the JAK-STAT signaling pathway,Toll-like receptor signaling and other immune-related pathways were significantly upregulated.Y.ruckeri mainly promote pathogenesis through the flagellum gene fli C in channel catfish.The weighted gene co-expression network analysis(WGCNA)R package was used to reveal that the infection of catfish is closely related to metabolic pathways.This study contributes to the understanding of the hostpathogen interaction between channel catfish and Y.ruckeri,more specifically how catfish respond to infection through a transcriptional perspective and how this infection leads to enteric red mouth disease(ERM)in these fish.2 Cloning and immune response of interleukin-22 from channel catfishThe molecular characteristics of the CDS gene sequence of I.punctatus interleukin-22(IL-22)and the response of IL-22 gene to different stimulate in gill,skin,hindgut,spleen,trunk kidney and head kidney immune organs and tissues as well as channel catfish kidney(CCK)cells of I.punctatus were explored.The results showed that the 534 bp CDS region of IL-22 was cloned and predicted to encode 177 amino acids.Multiple sequence alignment,Neighbor-Joining phylogenetic tree and gene homology analysis showed that it was highly conserved with vertebrates,and had the highest homology with Ameiurus melas.The results of quantitative real-time reverse transcription PCR(q RT-PCR)showed that IL-22 gene was widely distributed in 10 tissues of channel catfish,with the highest expression in hindgut and the lowest expression in heart.Lipopolysaccharide(LPS),Polyinosinic-polycytidylic acid(Poly(I :C)),Phytohaemagglutinin(PHA),Phorbol ester(PMA)stimulated channel catfish kidney cells(CCK)for 24-48 h.IL-22 was up-regulated under Poly(I:C)and LPS stimulation,and down-regulated under PHA and PMA stimulation.The injection experiments of Y.ruckeri,Streptococcus iniae,Channel catfish virus(CCV)and deltamethrin immersion experiments showed that the expression levels of IL-22 in gill,skin,hindgut,spleen,trunk kidney and head kidney were up-regulated as a whole.In the injection experiments of S.iniae and deltamethrin immersion experiments,the expression levels of IL-22 in gill were down-regulated.In the injection experiments of Y.ruckeri and CCV,the expression levels of IL-22 in skin were down-regulated.The above results showed that IL-22 gene expression patterns in different tissues were different under different stimulate.It is speculated that IL-22 gene of channel catfish can participate in a variety of immune responses and play a key role,which provided a reference for the further study of immune genes of channel catfish.3 Cloning and immune response of interleukin-10 from channel catfishIn this study,we cloned the predicted sequence of IL-10 gene of I.punctatus based on Nucleotide database.By comparing the sequence characteristics of IL-10 between different species,we constructed a NJ tree to evaluate the expression difference of IL-10 gene in different tissues of healthy I.punctatus individuals.And the response of I.punctatus CCK in vitro and in vivo under different stimulate.The results showed that672 base groups formed an open reading frame of IL-10 gene,encoding 223 amino acid residues,and the first 20 amino acid residues constituted a signal peptide.It contains six α-helixes and four conserved cysteine residues,which are Cys-31,Cys-81,Cys-130 and Cys-136,respectively.QRT-PCR showed that IL-10 gene was widely expressed in the tissues of healthy individuals of I.punctatus,with the highest expression in the liver.Poly(I:C),LPS,PHA,PMA stimulation of channel catfish kidney cells,24 h expression almost no change,48 h IL-10 expression showed varying degrees of increasing.In vivo injection of Y.ruckeri showed that the expression levels of spleen,trunk kidney and head kidney were up-regulated at 6 h after infection,and the expression levels of gill,skin,hindgut,spleen,trunk kidney and head kidney were significantly decreased at 6-48 h after infection.The expression levels of S.iniae,channel catfish virus injection and deltamethrin immersion were up-regulated after 6-48 h.Studies had shown that IL-10 may be involved in the body’s immune response to external stimulate.4 Cloning and immune response of interleukin-6 from channel catfishThe CDS region of IL-6 was successfully cloned by PCR and its sequence was analyzed.In addition,the expression levels of IL-6 gene in the gill,skin,hindgut,spleen,trunk kidney and head kidney of channel catfish and CCK under different stimulate were detected by q RT-PCR.The results showed that the CDS region of IL-6 was 642 bp,encoding 213 amino acids with isoelectric point of 7.75.Compared with mammals and fish,channel catfish IL-6 has the characteristics of sequence similarity,high collinearity and high conservation.This sequence contained the IL-6 / G-CSF / MGF family marker characteristic sequence(C-X(9)-C-X(6)-G-L-X(2)-Y-X(3)-L).NJ tree analysis showed that the sequence was highly similar to IL-6 of Ameiurus melas,clustered with fish first,and then clustered with mammals.The results of q-RTPCR showed that IL-6 was widely distributed in channel catfish,mainly in the spleen,followed by the heart and gonad,and the skin and liver were the least.Poly(I:C),LPS,PHA and PMA stimulated channel catfish kidney cells.CCK expression was significantly increased under PHA and PMA stimulation at 24 h,and IL-6 expression was increased at 48 h.In vitro injection of Y.ruckeri,S.iniae,channel catfish virus experiment and deltamethrin immersion channel catfish experiment showed that the expression of IL-6 in gill,skin,hindgut,spleen,trunk kidney and head kidney was upregulated,and the expression of IL-6 in gill was down-regulated in deltamethrin immersion experiment.In conclusion,the differential expression of IL-6 gene under different stimulation modes shows that IL-6 gene played an important role in the inflammatory response and immune response of gill,skin,hindgut,spleen,trunk kidney and head kidney of channel catfish,which can provide reference for the study of immune genes related to channel catfish.In summary,after acute infection with Y.ruckeri,strong antagonism between pathogens and host occurred.The CDS sequences of IL-22,IL-10 and IL-6 in I.punctatus are conserved,and have high homology with other species.Under different microbial stress,the expression level was up-regulated and involved in the immune response process.The above studies can provide basic data for the healthy cultivation of channel catfish from the perspective of immune prevention and control. |
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