Gene Cloning And Molecular Evolutionary Analysis Of Three Innate Immunity Genes Of Miiuy Croaker

Fish is a group of primitive vertebrates, their adaptive immunity is much lessadvanced than mammals. For the reasons of huge kinds and numbers of pathogens in theaquatic environment and less advanced adaptive immunity of fish, it is believed that theinnate immunity of fish will play more important roles in defending pathogen invasion.Three genes involved in innate immunity from miiuy croaker (Miithchys miiuy), includingthe third complement component (C3), the ninth complement component (C9) and theToll-like receptor2(TLR2), were cloned and analyzed by bioinformatics method. Theirexpression patterns of the healthy samples and those under the pathogen stress wereanalyzed by RT-PCR. And the molecular evolutionary analysis were conducted to explorewhether the huge differences between the aquatic and terrestrial environments, in whichthe teleost and mammals lived, had caused the differences on their evolutionary patternsand to research whether the innate genes of fish had undergone the positive selectionpressure for their less advanced adaptive immunity.The branch-site models detected twelve, five, four, and six positively selected sites onthe ancestral lineages leading to the vertebrates, the mammals, the ostariophysian and theprotacanthopterygian fish, and the actinopterygian fish, respectively, indicating that the C3genes had undergone at least four times of positive selection events. The vertebrates hadevolved the antibody and thus the classic pathway of complement system. The mammalshad evolved the complex and precision regulatory network of the complement system. Andthe fish had evolved the multiple forms of C3genes. The site models detected seven andzero positively selected sites in fish and mammals, respectively, indicating that themammals and the fish had experienced different evolutionary patterns, purifying selectionpressure for mammals and positive selection pressure for fish.A truncated form of C9molecular was cloned from miiuy croaker. It only presentedthree domains, namely TSP1, LDLRA and MACPF, which were located on the N terminal.Both of the EGF and TSP2domains on the C terminal were absent from the miiuy croakerC9, which is the first time of cloning a truncated C9form in teleost. Pathogen infectionexperiment demonstrated that the liver expressed thousand-fold of C9transcripts thanother tissues, demonstrating that the liver was the main resource of C9. And the expressionof C9in the kidney up-regulated fifty times than the control group, indicating that thekidney was the most changeable tissue for miiuy croaker during infection. Different with the C3genes, C9of fishes are under purifying selection pressure while the mammalian C9genes are under positive selection pressure for the reason that none positively selected siteswere detected in fish C9while six such sites were detected in mammalian C9genes.The TLR2gene of miiuy croaker is5135bp in length and is comprised of twelveexons and eleven introns. While both of the tiger fugu and pufferfish have only elevenexons and ten introns. Through comparing the numbers and locations of introns of TLR2genes between miiuy croaker and other fish, a direct evidence for intron insertionhypothesis on fish TLR2showed up. And the pathogen infection experiment indicated thatthe kidney was the main responder of TLR2in miiuy croaker for infection of Gramnegative bacteria. Both of mammalian and fish TLR2genes are under strong purifyingselection pressure.