Identification And Expression Analysis Of Three Genes Involved In Complement System From Grass Carp

The grass carp,Ctenopharyngodon idella,is a native Chinese freshwater fish with broad distribution from the natural water bodies of various basins in north and south China.Due to its good-quality meat,excellent growth performance,and wide adaptability,the grass carp has been transplanted to the rest of the world.According to Food and Agriculture Organization(FAO),the global grass carp production reached about 6.1 million tons in 2016.However,behind the rapid development of grass carp farming,high disease incidence and mortality due to bacterial septicemia has become a serious concern,and is mainly caused by Aeromonas hydrophila.Complement is considered as an important part of the innate immune system,which can play an important role in protecting the host from foreign pathogens.In this paper,A.hydrophila was used as the mediating factor to study and analyze three complement related genes(CFD,C4 and C3)of grass carp.The results are as follows:1.Functional study on the grass carp complement factor D(gcCFD)gene:The RACE method was used to obtain the full-length cDNA of gcCFD gene with a length of 935 bp,containing 753 bp open reading frame(ORF),encoding 250 amino acids.Through comparative analysis,it was found that gcCFD is a relatively conservative complement protein,which has a high similarity with other bony fishes.By constructing phylogenetic trees,it was found that the amino acid sequence of gcCFD was clustered together with other bony fishes,which supported the traditional evolutionary theory.gcCFD gene was expressed to varying degrees in twelve tissues of healthy grass carp,among which the expression level in the liver was the highest.After A.hydrophila infection,the expression level of gcCFD gene in six immune tissues were significantly upregulate.LPS,flagellin and A.hydrophila were used to stimulate hepatocytes,and it was found that all three factors could induce the expression of gcCFD gene.It was found that the liver cells with overexpression of gcCFD could significantly upregulate the expression of complement component 3(C3)gene and immune-related factors(such as IL-1?,TNF-? and IFN),and significantly increase the activity of NF-?B.To sum up,gcCFD gene plays a crucial role in the immune response of grass carps.2.Functional study on the grass carp complement C4(gcC4)gene:The RACE method was used to obtain the full-length cDNA of gcCFD gene with a length of 5458 bp,containing an open reading frame(ORF)of 5148 bp,encoding 1715 amino acids.Through comparative analysis,it was found that gcC4 was a relatively conservative complement protein and had a high similarity with other teleosts.By constructing phylogenetic tree,it was found that the amino acid sequence of gcC4 was clustered together with other teleosts,which supported the traditional evolutionary theory.gcC4 gene was expressed to varying degrees in twelve tissues of healthy grass carp,among which the expression level in the liver was the highest.During the bacterial infection,the expression level of gcC4 gene in six immune tissues changed to different degrees.The levels of C4 protein in the liver and serum were detected by Western blot and ELISA assay.Immunohistochemistry assay showed that the expression of C4 protein in the liver changed,and the expression of C4 in the hepatic necrotic area and hepatic sinus increased significantly after infection.LPS,flagellin and A.hydrophila were used to stimulate hepatocytes,and it was found that all three factors could induce the expression of gcC4.It was found that the liver cells with overexpression of gcC4 could significantly inhibit bacterial infection and proliferation,significantly upregulate the expression of C3 gene and immune-related factors(such as IL-1?,TNF-? and IFN),and significantly increase the activity of NF-?B.To sum up,gcC4 gene plays a crucial role in the immune response of grass carps.3.Functional study on the grass carp complement C3(gcC3)gene:gcC3 gene was expressed to varying degrees in twelve tissues of healthy grass carp,among which the expression level in the liver was the highest.During the bacterial infection,the expression level of gcC3 gene in six immune tissues changed to different degrees.The levels of C3 protein in the liver and serum were also detected by ELISA assay.Through histological analysis,it was found that liver damage and lesions occurred to different degrees during the infection process,and semi-quantitative systematic evaluation showed that liver damage was the most serious at 24 h.By immunohistochemistry assay,the expression sites of C3 protein in the liver migrated,and the expression of C3 in the hepatic necrotic area and hepatic sinus increased significantly after 24 h.To sum up,gcC3 plays an important immune role in grass carp resistance to A.hydrophila infection,and C3 can play an active role in repairing damaged liver tissue.