Bioinformatics And Expression Analysis Of MHC Ⅰ Region In Grass Carp

Grass carp（Ctenopharyngodon Idella）is one of the four major Chinese domestic carps and is also an important economic freshwater aquaculture fish.But in the culture process due to the impact of various diseases,its survival rate is very low and bring serious economic losses to the farmers.The bacterial septicemia caused by Aeromonas hydrophila is one of the diseases which is serious harmful to the health of grass carp in aquaculture.The major histocompatibility complex（MHC）,including many different genes,exists in all vertebrates and is an important part of antigen presentation involved in immune response.In this paper,the genomic information of the MHC Ⅰ region of the grass carp was obtained by local Blast,and was also compared with human MHC and the MHC Ⅰ region of other fish at the genomic level.It was helpful for evolutionary analysis by understanding the commonalities of MHC in different species.The expression of 9genes in the MHC Ⅰ region in grass carp was detected by semi-quantitative RT-PCR and the expression level of 4 immune-related MHC Ⅰ genes were detected in the spleen and kidney at 4,12,24,48 and 96 h after Aeromonas hydrophila infection by quantitative real-time PCR（q RT-PCR）.These results would provide a theoretical basis for the breeding of grass carp.The main results are as follows:1.The genomic data of the grass carp MHC Ⅰ regionThe results showed that there were 21 genes in the grass carp MHC Ⅰ region with a total length of 350 Kb,including three MHC Ⅰa genes,five PSMB genes,three TAP2 genes,two TAPBP genes and eight other genes including BRD2,FABGL,COL11A2,RXRB,KNSL2,ZBTB22,DAXX,FLOT1.2.Comparative analysis of the grass carp MHC Ⅰ region with other speciesComparative analysis of the data from human,Atlantic salmon,grass carp,medaka,zebrafish and fugu suggested that there were two sets of conserved gene blocks,which comprising comprised of the human extended class Ⅱ region and the classical class Ⅱ region,which in fish were linked to the MHC class Ia genes.The first conserved syteny consisted of the proteasome genes,such as PSMB8,PSMB10,PSMB9,TAP2,FABGL,BRD2,COLL11A2,and RXRB.The other conserved block of four to five genes was syntenic with the human extended class II region（including TAPBP,DAXX,ZBTB22,KNSL2 and,potentially,FLOT1 genes）.3.Alignment of the deduced grass carp TAPBP amino acid sequenceThe 396 amino acid sequence of grass carp TAPBP was obtained from NCBI.The alignment of grass carp TAPBP with TAPBP molecules of other vertebrates revealed the presence of the conserved Ig V and Ig C domains and two conserved cysteine residues at positions 31 and 106.Canonical cysteine and tryptophan residues(C¹⁹²、W²¹¹、C²⁷⁰和 C³¹⁴、W³²⁷、C³⁷⁸)were found in the predicted Ig V and C domains.Interestingly,the cysteine at positions 115 in human is replaced with a serine residue in avian and fish TAPBP.The c DNA sequence of grass carp PSMB9 was translated to 190 amino acids,of which 1-178 amino acids were Proteasome protein domains,and this region was highly conserved in fish,mammals and amphibians.The 342 amino acids of the grass carp MHC Ⅰ were obtained in the NCBI,among which 1-16 was the signal peptide,the MHC Ⅰ protein domain was located at position 17-192,the IGc1 functional region was 208-281,the297-319 position was the protein area.The evolutionary analysis of TAPBP,PSMB9 and MHC Ⅰ among MEGA7.0 found that the relationship between grass carp and Cyprinidae was the closest to other fish and farthest from other vertebrates.4.Gene expression patterns of genes in the MHC Ⅰ region in grass carpGene expression levels of BRD2,KNSL2,RXRB,TAPBP,FABGL,MHC Ⅰa,PSMB6,PSMB9 and TAP2 in various healthy tissues were detected by semi-quantitative RT-PCR.Except that FABGL and PSMB6 genes in the kidney had the highest expression,the other genes were highest expressed in the blood.The expression pattern of the TAPBP,MHC Ⅰa,PSMB9 and TAP2 genes which involved in the MHC class I antigen-presenting pathway were similar,and they were all highly expressed in the immune-related tissue such as the spleen and kidney.5.Expression of the immune-related BRD2,MHC Ⅰa,PSMB9 and TAP2 genes in the MHC Ⅰ region after bacterial infectionThe real-time fluorescence quantitative PCR was used to detect the expression offour immune-related genes in the grass carp MHC Ⅰ region after infection with A.hydrophila.The expression level of BRD2 gene reached the peak at 12 h in the kidney but24 h in the spleen.The expression level of MHC Ⅰa gene increased first and then decreased both in the spleen and kidney,and reached the maximum at 24 h.In the kidney,the expression of PSMB9 increased significantly and reached the peak at 12 h,and then decreased significantly at 48 h,following by a recovery to normal level at 96 h.In the kidney,the expression of PSMB9 peaked at 24 h,and then continued to decline.The expression levels of TAP2 gene were first down regulated and then up regulated in the spleen and kidney,and backed to normal level at last.These results indicated that genes in the MHC Ⅰ region of grass carp are related to the immune respose,laying a foundation for further research of controlling fish diseases and breeding resistance cultivars.