Molecular Characterization And Spatial-temperal Expression Of MHCⅡβ And GILT Genes Of Trachinotus Ovatus And The Correlation Analysis Between Their Polymorphic Sites And Resistant To Bacteria

In order to explore the mechanism of immunoregulation of Trachinotus ovatus,we obtained the partial sequence of MHC(major histocompatibility complex)and ToMHCⅡβ gene,and cloned the MHCⅡβ,GILT,gene by RACE technique.(ORF)747 bp,encoding 248 amino acid.The phylogenetic tree analysis constructed by NJ method showed that all Trachinotus ovatus were clustered together,which were close to the other fishes but distant from amphibians and mammals.QRT-PCR analysis showed that MHCⅡβ mRNA was expressed in 13 tissues and had higher expression levels in spleen and head kidney,and the lowest expression in fins.The expression of MHCⅡβ mRNA was significantly up-regulated in the liver,spleen,head kidney and intestine after challenged with Photobacterium damselae.In this study,nucleotide polymorphism analysis showed that base deletion,shift and insertion mutation weren’t found in the 278 bp sequences,and these sequences found a total of 69 nucleotide mutation sites(the mutation rate is 24.8%),which exist 6 dinucleotide mutations,7 trinucleotide mutations,and 2 tetranucleotide mutations.The entire nucleotide mutation are distributed from 18 to 50 bp.It was found that the 72 amino acids with 28 cite were polymorphic with a mutation rate of 38.29%.The non-synonymous mutation rate(dN),synonymous mutation rate(dS)of the susceptible population and the resistance group sequence and their ratio(w = dN / dS)are shown in Table 3.Statistical analysis of frequency distribution to these obtained gene bands in susceptible and susceptible groups showed that 6 bands appeared in the resistance group and 13 bands appeared in the susceptible group.Finally,6 bands were selected as the next statistical analysis,and their distribution in the two groups were analyzed significantly.The results showed that the distribution frequency(0.223)of band TO-DAB-01 in resistance group was significantly higher than susceptible group(0.058)(P<0.01)04,TO-DAB-05.TO-DAB-10 in the susceptible population(0.045,0.120,0.063)were significantly higher than resistance group(0.008,0.050,0.020)(P<0.01).While the distribution frequency of the other two bands did not show any significant differences between the susceptible group and the susceptible group.Therefore,it was concluded that the band of TO-DAB-01 is associated with the resistance to Trachinotus ovatus,while the bands of TO-DAB-04,TO-DAB-05,and TO-DAB-10 showed susceptibility to mermaid bacilli.In order to investigate MHCⅡβ cite in the genome,five positive clones in each individual were analyzed.The results showed that the number of detected haplotypes of individual were from 2 to 3,Suggesting that there are more than two MHCIIβ gene cites exist in the Trachinotus ovatus genome.The DNA length of GILT gene was 6837 bp,and includes 232 bp 5 ’untranslated region(UTR),771 bp ORF and 1746 bp 3’ untranslated region(3’UTR).The GILT gene encodes 256 amino acids and a theoretical relative molecular weight is 28.6 kD and theoretical isoelectric point is 5.97.Using the method of bioinformatics,we can understand the structure of two genes.The results showed that ToGILT mRNA was expressed in 13 tissues.There was a significant difference between the expression of liver and head kidney(P<0.05).The same infection of the mermaid hair bacilli,the expression of ToGILT mRNA in the liver,spleen,head kidney,intestine were up-regulated.The expression level of the spleen and the head kidney began to increase,and the expression level of the spleen and head kidney tissue was up-regulated at 12 h,and then began to decrease,and back to the initial expression at 72 h.There was a SNP site in the ToGILT gene,in which the distribution frequency of genotype CG in the susceptible population was significantly higher than the susceptible population(0.148)(P<0.01)The distribution frequencyin the susceptible population(0.612)was significantly higher than that in the susceptible population(0.388)(P<0.05).This study explored the immune regulation mechanism in Trachinotus ovatus and lay the foundation for further research in the future.