Screen Of Antibacterial Immune Genes And Function Analysis Of Lectin Pathway In Grass Carp

The grass carp(Ctenopharyngodon idella)is a dominant commercially farmed species.In the present thesis,high throughput sequencing was used to determine different expressed immune-related genes and analyze the function mannose-binding lectin(MBL),mannan-binding lectin-associate protease 1(MASP-1)and complement component 2(C2)in innate immunity of grass carp.The contents and results of this study are as follows: 1.de novo RNA-Seq sequencing of spleen tissueIn the present study,we performed de novo RNA-Seq sequencing of spleen tissue from grass carp,which were given intra-peritoneal injections containing PBS with or without a dose of A.hydrophila.The fish were sampled from the control group at 0h,and from the experimental group at 4,8,12,24,48 and 72 h.149,514,670 raw reads were obtained from 7 cDNA databases.After fliter,122,181,346 clean reads,total of 42.67 G data,were obtained from the normalized cDNA libraries;these were assembled into 191,795 transcripts.Of those,2,992 genes were used in the analysis of differentially expressed genes(DEGs).The results of analysis DEGs and qRT-PCR revealed that:(1)Complement components function a vital role in the defense against A.hydrophila infecting grass carps.(2)The complement system was activated at 4 h following A.hydrophila challenge.(3)The transcriptional levels were firstly increased then decreased,which indicated complement system may involve in signal transmission.2.MBL mediates immune responses to A.hydrophila infectionMBL,the initiating molecule of the lectin pathway,plays a significant role in the innate immune system in mammals and fish.The full-length 948 bp gcMBL cDNA includes a 741 bp open reading frame encoding a 246 amino acid protein.Transcription of gcMBL was widely distributed in different tissues,and was induced by A.hydrophila in vivo and in vitro.Expression of gcMBL was also affected by LPS and flagellin stimulation in vitro.In cells over-expressing gcMBL,transcripts of almost all components except gcC5 were significantly enhanced,and gcMBL,gcIL1?,gcTNF-?,gcIFN,gcCD59,gcC5 aR and gcITG?-2 were significantly up-regulated following exposure to A.hydrophila or stimulation by bacterial PAMPs.Meanwhile,gcMBL interference down-regulated transcript levels following A.hydrophila challenge,and gcMBL activated NF-?B signalling.These findings indicate a vital role for gcMBL in innate immunity in C.idella.3.MASP-1 mediates immune responses to A.hydrophila infectionMASP-1 gene is a crucial component in lectin pathway of the complement and coagulation cascade.The full-length 3308 bp gcMASP-1 cDNA includes a 2160 bp ORF.Transcription of gcMASP-1 was widely distributed in different tissues,and was induced by Aeromonas hydrophila in vivo and in vitro.Expression of gcMASP-1 was also affected by LPS and flagellin stimulation in vitro.In cells over-expressing gcMASP-1,transcripts of almost all components except gcMBL and gcC5 were significantly enhanced,and gcIL1?,gcTNF-?,gcIFN,gcCD59,gcC5aR1 and gcITG?-2 were significantly up-regulated following exposure to A.hydrophila.Meanwhile,gcMASP-1 interference down-regulated transcript levels following A.hydrophila challenge,and gcMASP-1 activated NF-?B signalling.These findings indicate a vital role for gcMASP-1 in innate immunity in C.idella.4.C2 mediates immune responses to A.hydrophila infectionC2 is an important component of C3 convertase.gcC2 transcription was widely distributed in different tissues,induced by Aeromonas hydrophila in vivo and in vitro,and affected by lipopolysaccharide and flagellin stimulation in vitro.In cells over-expressing gcC2,transcript levels of all components except gcC5 were significantly enhanced,and gcC2,gcIL1?,gcTNF-?,gcIFN,gcCD59,gcC5aR1,and gcITG?-2 were significantly upregulated after A.hydrophila challenge or stimulation with bacterial pathogen-associated molecular patterns(PAMPs).However,gcC2 in interference cells down-regulated the transcript levels after A.hydrophila challenge,and gcC2 activated NF-?B signaling.These findings indicate the vital role of gcC2 in innate immunity in grass carp.