| Megalobrama amblycephala is one of the important herbivorous economic fishes in China.In 2015,the production of blunthead bream has exceeded 500000 tons.At present,the production of blunthead bream in Jiangsu Province accounts for a quarter of the national production.With the continuous development of blunt snout bream farming,intensive and high-density farming methods lead to disease outbreak and even a large number of deaths,which restricts the sustainable development of blunt snout bream farming.In the outbreak of disease,the immune system is the primary defense line against pathogenic microorganisms in fish,which plays an important role in the complement system and inflammatory response.Complement is widely involved in the mechanism of immunoregulation,which has the function of immunoregulation and mediating immunopathology.As an indicator of inflammation,interleukin acts as an intercellular medium to regulate the intensity and duration of immune response.Therefore,the study of complement system and inflammatory response of Megalobrama amblycephala lays a theoretical foundation for revealing the response mechanism of environmental stress.At present,the laboratory has successively carried out the research on the construction of nutrition requirement database,the screening of high-quality feed protein source,nutrition and immune regulation,etc.,but the research on the interaction between nutrition and environment is relatively limited,especially the immune response mechanism of blunt snout bream to food deficiency and bacterial infection is rarely reported.In this paper,the mechanism of complement system response of Megalobrama amblycephala fed after starvation was studied.By analyzing the expression process of C3 and C9 genes,the regulatory effect of complement on the growth compensation effect in starvation was explained.The tissue changes of liver and intestine were observed to clarify the reversible point of tissue structure under starvation stress.Aeromonas hydrophila,as one of the main pathogens of freshwater fish,greatly affects the healthy culture of Megalobrama amblycephala.The purpose of this study was to investigate the changes of IL gene expression of Megalobrama amblycephala after infection with Aeromonas hydrophila to study the regulatory effect and inflammatory response of inflammatory genes on anti infection,and to verify the histopathological changes of Megalobrama amblycephala after infection with Aeromonas hydrophila by observing the ultrastructure of liver tissue.The main content mainly includes the following tw o aspects of research:1.The full-length cDNA sequences of C3 and C9 genes(MaC3 and MaC9)were cloned from Megalobrama amblycephala cDNA library by race technology for the first time.The expression of MaC3 and MaC9 genes in different tissues of Megalobrama amblycephala was detected by qRT-PCR and the expression patterns in liver after starvation stress were analyzed.The results showed that the total length of MaC3 and MaC9 genes was 5378 bp and 2522 bp,respectively,the length of 5’UTR was 316 bp and 8 bp,the length of 3’UTR was 133 bp and 480 bp,the length of open reading frame was 4929 bp and 2034 bp,respectively,encoding 1642 and 677 amino acids.The homology analysis of amino acid sequence showed that the similarity of MaC3 and MaC9 with Carassius auratus complement C3 and Ctenopharyngodon idella complement C9 of Carassius auratus and Ctenopharyngodon idella were 78%and 84%,respectively.The results of real-time fluorescence quantitative analysis showed that MaC3 and MaC9 were expressed in brain,liver,spleen,head kidney,middle kidney,heart,foregut,midgut,hindgut,gill and muscle tissues,with the highest expression in liver and the lowest expression in kidney and heart respectively.Four groups were designed:(1)normal control group:3%of body weight was fed with experimental diet daily for 28 days;(2)starvation and re feeding group:1-14 days of starvation,15-28 days of daily feeding according to 3%of body weight,no surplus feed within 30 minutes;(3)overfeeding group:1-14 days of starvation,15-28 days of daily feeding according to 8%of body weight,30 There was still surplus feed after min;(4)starvation group:no feed during the experiment,culture cycle 28 days.On the 21st and 28th day of the experimental period,the expression of C3 and C9 genes in the liver of the starving group was significantly lower than that in the control group(P<0.05),but the expression of C3 and C9 genes in the starving group was significantly higher than that in the control group(P<0.05),and the expression of C3 and C9 genes in the starving group was not significantly different from that in the control group.In the group of persistent starvation,the nuclei of the hepatocytes of Megalobrama amblycephala were blurred and deformed,and the sinuses of the liver blood were dilated.There was no significant difference between the starvation group and the control group,the lamina propria was slightly expanded,and leukocyte infiltration appeared in the epithelium of the starvation group.2.The full-length cDNA sequences of Megalobrama amblycephala IL1β and 1L1R genes were cloned by race technology,and named as MaIL-1β and MaIL-1R.The expression of MaIL-1β and MaIL-1R genes in different tissues of Megalobrama and in liver tissues of Megalobrama amblycephala after infection with Aeromonas hydrophila were detected by qRT-PCR.The total length of the cDNA sequence of MaIL-1β gene is 1165bp,the length of coding region is 729bp,242 amino acids are encoded,5’UTR is 56bp,3’UTR is 380bp.The total length of the cDNA sequence of MaIL-1R gene is 2026 BP,the ORF region is 1068 BP,encoding 535 amino acids,the 5’untranslated region is 169 bp,and the 3’untranslated region is 249 bp.The amino acid sequences of IL-1β and IL-1R genes were compared and analyzed.Among them,the similarity of IL-1β gene with Anabarilius grahami was as high as 88.4%,while the similarity of IL-1β gene with other species was between 59.2%and 76.4%.The highest homology with the IL-1β gene of Megalobrama amblycephala was found in whitefish(86.4%).The homology with the IL-1R gene of other species ranged from 38.3%to 74.1%.IL-1β and IL-1R genes are expressed in brain,liver,spleen,head kidney,middle kidney,heart,foregut,midgut,hindgut,gill and muscle.The expression of IL-1β was the highest in the foregut,higher in the head kidney and spleen,and lower in the brain,middle kidney and middle intestine.The expression of IL-1R gene was the highest in spleen,and also in liver,middle kidney and heart.The experimental design is that the concentration of Aeromonas hydrophila in the experimental group is 1*10^6 CFU/ml,and the injection volume is 1%of the body weight;the control group is injected with 0.65%sterile saline of the same volume.Samples were taken at 3h,6h,12h,24h and 48h respectively.The expression level of IL-1β gene and IL-1R gene increased first and then decreased,and reached the highest level 12 hours after the virus attack.With the prolongation of the time of attack,the nucleus of liver cells gradually appeared fuzzy deformation and inflammatory cell infiltration.In addition,the mitochondria of hepatocytes were slightly swollen,and the number of mitochondria in spinal blur was reduced and swollen. |
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