The Sequence Analysis And Detection Of Cyt B Gene In Trachidermus Fasciatus And MyD88Gene In Lateolabrax Japonicus

The mitochondrial cytochrome b（Cyt b）gene of Trachidermus fasciatus Heckeland myeloid differentiation factor88（MyD88） gene of Lateolabrax japonicus werecloned. In particular,the former was amplified by universal PCR and the latter wasamplified by rapid-amplification of cDNA （RACE） PCR. Then fluorogenicquantitative PCR method were established for detection of Cyt b and MyD88gene.The complete sequence of Cyt b is1141bp in Trachidermus fasciatus Heckel.The average percentage of T,C,A,G is28.2%,32.0%,22.9%,16.8%. The third codonexhibits anti-G bias，which is a common characteristics of vertebrates. The π value ofTajima’s neutral test equals to0.4674%. The Sequence comparison between10species of Cottidae and Trachidermus fasciatus Heckel has been carried out by Mega4.0software for phylogentic trees and analysis of sequence distance. It is showedthat Trachidermus fasciatus Heckel is clustered to a single clade. There is relativelyfarther relationship between Laterolabrax japonicus and Trachidermus fasciatusHeckel. The quantitative PCR amplification with good sensitivity and speciality hasbeen established for detection Cyt b gene of Trachidermus fasciatus Heckel. Thedetecting limit of this method is3.20×102copies/reaction. This work lays thegroundwork of research for molecular ecology and genetic diversity and benefits forprotection of Trachidermus fasciatus Heckel.The full-length of MyD88cDNA is1739bp in Lateolabrax japonicus. Thecloned cDNA exhibits105bp of5’-UTR,931bp of3’-UTR and903bp of the entireopen reading frame (ORF). The ORF encode a polypeptide of300amino acids,which has molecular mass of34.02kDa. There is a Polyadenylation signal ATTAAAin the lower reaches of3’-UTR. The ORF contains two domains, called deathdomain(DD) with80amino acids and TIR domain with144amino acids. The result of comparing MyD88gene of Lateolabrax japonicus,some fishes and Mus musculusis that the TIR domain is more conservative than DD. In common, the identitypercentage of TIR domain is greater than80%, but DD is between60%and80%.Three conserved cDNA regions（box1、box2、box3） are found by comparing aminoacid sequences. The phylogenetic tree of MyD88gene constructed by MegAlignsoftware is basically in line with the classification of each species. But, two branchesget low confidence level and are irresponsible. It’s considered that MyD88gene withgood conservatism is unsuited to analyse relationship. The relative fluorescentquantitative PCR method has been founded. The reference gene is18S and bladder iscontrol tissue. Using the method,the MyD88gene expression of some organs andtissues were analysed in Lateolabrax japonicus. It’s indicated that MyD88expressedin all organs and tissues，especially spleen,oral epithelium and gills. The relativefluorescent quantitative PCR method is preparation for research of the immunmechanism of MyD88of fish.