Genetic Screening、cloning And Expressing And Immune Response Of Immune-Related Gene Of Pelodiscus Sinensis

With the development of intensive farming,the breeding environment is deteriorating and the disease is frequent.The sustainable development of the Chinese soft-shelled turtle breeding industry is considerably constrained.In order to explore the Chinese soft-shelled turtle response process against bacteria at the transcriptomic level,this study used Illumina HiSeqTM 2500 high-throughput sequencing technology to examined transcriptional profiles on Chinese soft-shelled turtle spleen.According to the results,MHCⅡα and MMR1-LIKE were selected for analyzing cloning and expressing and immune response.The results can be summarized as follows:1.RNA-sequencing and screening of immune related genesAs a result,48 884 unigenes were acquired after assembly and redundancy.Comparing unigenes with COG,GO,KEGG,KOG,Pfam,Swissprot,eggNOG and NR databases,a total of 17 202 annotations were obtained.The Chinese soft-shelled turtle injected A.hydrophila group was regarded as the experimental group,and the Chinese soft-shelled turtle injected PBS group was set as the control group to screen differentially expressed genes,the results showed that there were 2 300 Unigenes differentially expressed,including899 up-regulated genes and 1 401 down-regulated genes.Moreover,the differentially expressed genes were categorized by genomes(KEGG)enrichment analysis,the results revealed that there were 3 immune-related pathways(Intestinal immune network for Ig A production pathway,Hematopoietic cell lineage pathway and Fc gamma R-mediated phagocytosis pathway)in the top 20 pathways that the differential genes were significantly enriched.Additionally,in light of KEGG and GO annotation,36 differentially expressed immune-related genes were screened and classified into eight categories according to function: Antigen processing and presentation,Innate immune molecules,T/B cell activation and proliferation,Toll-like receptor signaling pathway,Cell adhesion,immunosignal molecule,Complement activation,and Chemokines and chemokine receptors.After that 18 genes were randomly selected from the differentially expressed immune-related genes for qRT-PCR confirmation,and the results illustrated that the trend was consistent with the results of transcriptome sequencing analysis,indicating that the transcriptome data were accurate and reliable.2.Full-length cDNA cloning,expression and immune response of MHCⅡα and MMR1-LIKE genesTo study the structure and function of MHC(major histocompatibility complex)in Pelodiscus sinensis,we obtained the full-length cDNA of MHCⅡα using RACE-PCR(rapid amplification of cDNA ends PCR)technology.The resulting sequence was 1296 bp in length,including an ORF(open reading frame)region of 807 bp.The peptide-encoded MHCⅡαgene could be divided into four parts,including the signal peptide,MHCⅡ alpha domain,IGc1 domain,and transmembrane region.A neighbor-joining tree showed that the MHCⅡαgene from P.sinensis and Chrysemys picta bellii cluster into one cluster.The P.sinensis gene had a close genetic-relationship with C.picta bellii and a farther genetic-relationship with mammals,aves,and fish.Using qRT-PCR in all 8 tissues,the highest expression of the MHCⅡα gene was observed in the spleen,heart,liver,and intestine,while the lowest level was found in muscle.Meanwhile,MHCⅡα mRNA expression levels were significantly up-regulated in the liver and intestine(after 12 h),the spleen(on the 1st day),and the kidney(on 1st and 5th day)after being infected with Aeromonas hydrophila.The liver,spleen,intestine,and kidney are closely related to immunity,which indicates that this gene has important effects on the immune response.MR can assist macrophages to phagocytize and process glycoproteins,and present to MHC-Ⅱ,To study the structure and function of MR(mannose receptor)in Pelodiscus sinensis,we obtained the full-length cDNA of MMR1-LIKE using RACE-PCR(rapid amplification of cDNA ends PCR)technology.The resulting sequence was 1416 bp in length,including an ORF(open reading frame)region of 1137 bp.The peptide-encoded MMR1-LIKE gene could be divided into four parts,including the low complexity regions,Transmembrane region,RICIN,and CLECTs.A neighbor-joining tree showed that the MMR1-LIKE gene from P.sinensis,Terrapene carolina triunguis,Chrysemys picta bellii,and Chelonia mydas cluster into one cluster.The P.sinensis gene had a close genetic-relationship with T.carolina triunguis,C.picta bellii and C.picta bellii and a farther genetic-relationship with mammals,poultry,and fish.Using qRT-PCR in all 8 tissues,the highest expression of the MMR1-LIKE gene was observed in the liver,while the lowest level was found in intestine and stomach.Meanwhile,MMR1-LIKE mRNA expression levels were significantly up-regulated in the liver,the spleen,the intestine,and the kidney after being infected with A.hydrophila.The liver,spleen,intestine,and kidney are closely related to immunity,which indicates that this gene has important effects on the immune response.