| Hyriopsis cumingii(Hyriopsis cumingii)is one of the most important freshwater pearl mussel in China.With the continuous expansion of aquaculture scale,the occurrence of diseases is more and more frequent,causing great losses to the shellfish aquaculture industry in China.Notch signaling pathway is widely found in multicellular organisms and plays an important regulatory role in cell growth,development,differentiation,apoptosis,tissue remodeling,and inflammation.Notch signaling was initially identified as a pleiotropic regulator of cell fate,and has recently been implicated in immune cell development and function.In this study,hree Notch ligands were cloned and identified and their structural characteristics were analyzed.The tissue expression patterns and pathogen-related molecular patterns(lipopolysaccharide,peptidoglycan and polycytidylinic acid)were analyzed by real-time fluorescence quantitative analysis.These ligands were knocked down by RNAi technology to detect the expression changes of some immune signaling pathway genes and immune proteins in mussels.In addition,recombinant proteins were expressed in vitro and their interactions with bacteria were analyzed.The results of the study enriched the understanding of the innate immunity of the mussel,and provided a theoretical basis for healthy shellfish culture.1.Cloning and analysis of notch ligand genes in the musselThree Notch ligands were screened and cloned from the transcriptome library of H.cumingii,.The full-length c DNA of HcDelta like1,HcDelta like2,and Hc Jagged1 were 4117 bp,2128 bp and 3123 bp,respectively,and were predicted to encode 786,656,616 amino acids.Bioinformatics predicted that the HcDelta like 1protein contained a MNNL structure function domain,but HcDelta like2 and Hc Jagged1 did not..HcDelta like1,HcDelta like2,Hc Jagged1 all contained different numbers of EGF repeats;HcDelta like1 had 3 EGF,6 EGF-CA and 1 EGF-like structural domains;HcDelta like2 had 8 EGF and 2 EGF-CA;Hc Jagged1 had 2 EGF and 3 EGF-GA.Sequence alignment analysis showed that HcDelta like1 had the highest identities of 53.18% with Mediterranean mussel,HcDelta like2 had the highest identities of 43.22% with Haliotis rufescens and Hc Jagged1 had the highest identities of 43.37% with Crassostrea gigas.2.Notch ligand members are involved in the immune response of musselsReal-time quantitative PCR analysis showed that HcDelta like1,HcDelta like2,and Hcjagged1 were all expressed in the heart,intestine,kidney,muscle,mantle,gill,axe foot,and gonad of the mussel.HcDelta-like1 had the highest expression in the adductor muscle and the lowest expression in the heart;The expression of HCdeltalike2 was highest in intestine and lowest in adductor muscle;Hc Jagged1 expression was highest in the hepatopancreas and lowest in the axe footAfter LPS,PGN and Poly I:C stimulation,the m RNA expression levels of HcDelta like1,HcDelta like2 and Hcjagged1 in the hepatopancreas and gills of mussels generally showed a decreasing and then increasing trend.The results of RNA interference assay showed that after HcDelta like1 knockdown,the expression levels of immune-related genes Bcl-2 and NF-κB2 were significantly decreased,and the contents of HMGB-1 and TNF-α in hemocytes were significantly decreased.NFNF-κB2 expression was also significantly decreased after HcDelta like2 knockdown.These results suggested that Notch ligands are involved in the immune response of musselsThe extracellular domains of HcDelta like1 and HcDelta like2 were recombinant expressed in vitro,and the recombinant proteins had the ability to bind gram-positive and gram-negative bacteria,and could bind LPS,PGN and poly I:C.In situ hybridization of hemocytes showed that HcDelta-like1 and HcDeltalike2 were both expressed in hemocytes.The expression level of HcDelta-like2 decreased at 12 h after LPS and PGN injection and increased at 24 h after injection.HcDelta-like1 and HcDelta-like2 expression levels increased at 12 and 24 h after injection of poly(I:C). |
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