Cloning And Expression Analysis Of MHCII, MyD88 And TLR2 Gene CDNAs From The River Shatang

Odontobutis potamophila is a unique small freshwater demersal carnivorous fish in China.Current researches of O.potamophila mainly focus on the selective breeding and cultivation.Studies on the hereditary basis such as immune related genes are still relatively less.Therefore,it is important to conduct researches on the study of disease-resistant genes of immune system and parsing the immune mechanism,to control the diseases of O.potamophila and develop new approaches disease resistance breeding.In this study,rapid amplification of cDNA ends(RACE)approches were used to obtain the full length of MHC?A,MHC?B,MyD88 and TLR2 cDNA and quantitative real-time PCR were used to analysis the expression pattern.The main results were shown as follows:1.Sequence analysis revealed that the full length of MHC?A,MHC?B,MyD88 and TLR2 cDNA were 884 bp,896 bp,1445 bp and 3473 bp respectively.And the putative length of open reading frame(ORF)in the four genes were predicted as 711 bp,726 bp,726 bp,and 2631 bp that encoded 236,241,288,876 amino acids.The length of the 5' of the four genes were 19 bp,20 bp,31 bp,and 75 bp,and the length of 3' UTR were 154 bp,150 bp,557 bp,and 767 bp.Each 3' UTR in the four genes possesseda polyadenylation signal sequences of AATAAA,and a poly A tails of 32 bp,28 bp,26 bp and 30 bp,respectively.2.Bionemerics were used for analysisthe structure of the four genes,and results indicated that the two polypeptide chain encoded by MHC?A and MHC?B both included one signal peptide,two extracellular domains(?-1 and ?-2,?-1 and ?-2),one transmembrane domain and cytoplasmic domain.The structures had conservative cysteine residues and rich phosphorylation sites.The spatial structures of MHC?A and MHC?B were predicted,and the results indicated that both MHC?A and MHC?B had classical spatial structures of MHC? class.It was declared that both MHC?A and MHC?B were belongs to the MHC family.There were death domain and TIR domain in both N-terminal and C-terminal of MyD88.Secondary structure prediction indicated that many functional sites in the MyD88 protein molecule.TLR2 protein of O.potamophila had LRR motifs and TIR domain structure.This is an important characteristic of TLR family.In the TLR2 protein molecule,there also were many factional sites.3.Comparative sequence analyses indicated that consistencies of MHC?A,MHC?B,MyD88 and TLR2 genes in O.potamophila with other fish were 42-52%,37-52%,43-61%,and 48-63%,respectively.Phylogenetic trees were built according to the amino acid sequences of MHC?A,MHC?B,MyD88 and TLR2.Similar results were obtained that the four genes in O.potamophila had closed genetic relationship with Perciformes.4.Quantitative real-time PCR was used to quantify the expression of MHC?A,MHC?B,MyD88 and TLR2 genes in twelve(liver,blood,kidney,intestine,gill,heart,muscle,stomach,spleen,brain,gonad and swim bladder)tissues of healthy O.Potamophila.MHC?A expressed highly in spleen,intestine,and gill.MHC?B expressed highly in kidney,intestine and blood.MyD88 expressed highly in liver,kidney,gill and blood.TLR2 expressed highly in liver,kidney,intestine,blood and stomach.The four genes expressed moderately or low in other organs.The results declared that these four genes highly expressed in immune organs and tissues.5.The mRNA expression levels of MHC?A,MHC?B,MyD88 and TLR2 genes in liver,spleen and kidney of O.potamophila after Aeromonas hydrophila challenge were quantified by quantitative real-time PCR with ?-actin as internal control.The results showed that,MHC?A,MHC?B,MyD88 and TLR2 changed at mRNA level to certain degrees.The expression quantities of MHC?A in three major immune systems fluctuated as the infection continued,with similar change trend.The expression quantities dropped slightly 4 hours after injection.The expression quantity reached peak after 24 hours in the spleen,and then declined slightly from 48 to 96 hours.In the kidney and liver,the expression levels sustained a slight fluctuation,but several times higher than the level in the 4 hour.In the spleen and liver,the expression level of MHC?B at first ascended,and dropped,then reascended.The expression level reached peak at 96 hours and 4 hours in spleen and liver respectively.In the kidney,MHC?B expressed less after injection and reached the minimum at 24 hours,and then expressed higher afterwards.The expressions of MyD88 gene showed a consistent trend in the spleen and liver.MyD88 expressed higher in 4-96 hours than the level before injection.The expression level ascended higher in spleen than liver.In the nephridial tissue,however,the expression level dropped slightly in 4-24 hours after injection,and then ascended again and maintained a high level.The expression levels of TLR2 also changed after Aeromonas hydrophila challenge.It demonstrats that the four genes involved in the body's immune response to bacterial infection.