| In recent years,with the increasing density of cage culture,the breeding of large yellow croaker(Larimichthys crocea)is frequent,with a wide range of diseases and high mortality,which seriously restricts the economic interests of fishermen and people’s health.Our group used Vibro anguillarum and Vibrio Parahaemolyticus to infect juvenile large yellow croaker,recorded the number of deaths per day,and observed the site of the infected disease;selected the infected group on the seventh day of the live disease.The diseased rhubarb fry,and the control group were subjected to transcriptome sequencing;the SNP sites of the immunological differential genes were screened from the transcriptome and verified.Through the infection experiment,it was found that the mortality of the V.anguillarum infection group increased sharply from the third day;the mortality of the V.parahaemolyticus infection group increased sharply from the second day,and the mortality rate reached the maximum on the fifth day.Part of the large yellow croaker with the infection of V.anguillarum showed intracranial,temporal and abdominal hyperemia and sub-spinal hemorrhage.Morphological observation of the parasitic blood sample of V.parahaemolyticus showed that the symptoms were almost the same as those of V.parahaemolyticus,but there was no symptoms of sub-vertebral hemorrhage.Histopathological study found that the pathological samples of V.anguillarum and V.parahaemolyticus showed empty tissues such as brain,head kidney,sputum and subcutaneous mucosa.The intestines were black,the intestinal mucosa fell off,the liver was loose,and the kidneys showed more red blood cell symptoms.The pathological samples of V.anguillarum infection also found spinal cord congestion and more inflammatory cell symptoms.High-throughput sequencing of large yellow croakers before and after infection with V.anguillarum and V.parahaemolyticus was carried out using illumina Hi Seq TM sequencing technology.A total of 188,132,064 pairs of 90 reads of Raw reads were obtained.After data processing,a total of 183,375,780 pairs of Clean reads were obtained.Nearly 20,000 genes receive GO annotations,including cell components,biological processes,and molecular functions.Along with the genomic mapping results of all the data,a total of 13901 new transcripts were predicted.We screened 2136 immune-related genes from the transcriptome of V.anguillarum and V.parahaemolyticus.Among them,there were 140 differentially expressed genes(P<0.1)in the symptomatic group(ZFY)and the anti-V.parahaemolyticus group(ZFW)and V.anguillarum(ZMW),and the immune-related genes were Forty-five,there are 92up-regulated genes.The control group(ZWC)had 228 differentially expressed immune genes compared with V.anguillarum and V.parahaemolyticus(P<0.01),and the control group screened 101 differential immune genes compared with V.anguillarum.The anti-V.parahaemolyticus group screened 127 differential immune genes.Compared with ZMW,ZWC screened 101 immune differential genes,of which ZMW up-regulated 39genes and down-regulated genes 62.Among the 39 up-regulated genes,14 gene coding regions were screened for 64 SNP sites,and 17 of them had significant mutation sites leading to amino acid changes.In addition,ZWC found a total of 127 immune differential genes compared with ZFW.Among them,ZFW has 53 immune genes up-regulated,73 down-regulated genes,and 53 immune up-regulated genes found 29gene coding regions with 146 SNP loci,which lead to amino acid changes.There are 28meaningful SNP loci.The SNP site of some gene coding regions has been verified by SNP.It is found that the susceptible individuals of V.anguillarum are located on the chromosome NW＿011323507.1.The interleukin receptor 6 gene(IL-6R)has a mutation in the91196th base.The acid mutation was leucine.This site mutation was significantly different between the survival sample on the seventh day of V.anguillarum infection and the control sample(X~2=6.838),indicating that the mutation of the IL-6R gene at position 91196 was against the large yellow croaker.Anti-Vibrio disease is associated;the 35665 base mutation on the chromosome of the complement C1q/TNF-related protein 9(CTRP9)gene is extremely positive in the seventh day of survival of the V.parahaemolyticus infection group and the fourth day of death.Significant differences(X~2=11.879)indicate that the CTRP9-based site mutation may be associated with resistance to V.parahaemolyticus in large yellow croaker.The full-length c DNA sequence of Nitric oxide synthase(NOS1)was obtained from the transcriptome.PCR amplification and sequencing confirmed that the sequence was 4281 bp and the gene encoded 1426 amino acids.The phylogenetic tree was constructed to know that the NOS1 gene of large yellow croaker was clustered with NOS1 of the semi-smooth tongue,gingiva,red-fin oriental carp,and turbot NOS1,and the similarity of large yellow croaker and turbot was 82.81%.The NOS1 gene of large yellow croaker has a relatively long evolutionary relationship with zebrafish and golden locust,and has a farther evolution relationship with rats and mice,and has a farther relationship with humans and macaques.Eight SNPs were found in the full-length c DNA of NOS1 gene of large yellow croaker.The first generation of sequencing found that the 829 Pro mutation was Ala,which was a significant mutation.After sequencing,it was confirmed that the V.anguillarum infection group was significantly different from the control group(X~2=14.7).There was a significant difference in the V.parahaemolyticus infection group(X~2=8.3).In summary,the SNPs of the immune differential gene of V.anguillarum and V.parahaemolyticus infected with V.anguillarum were screened and verified for the first time,and three SNP loci against Vibrio infection were identified.The KEGG enrichment analysis found that the ZFW group and There was a big difference between the control group and the V.anguillarum infection group;these all lay a good foundation for the selection of large yellow croakers against V.anguillarum and V.parahaemolyticus resistant populations. |
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