| Roughskin sculpin,(Trachidermus fasciatus Heckel), is a kind of carnivorous fish, which has seawater-freshwater migratory habits. As one of the second order of protected animals in China, roughskin sculpin can be found only in Yalu River in the northeast, Qinglong River in Shandong Province and Fuchun River in Zhejiang Province nowadays. Due to the lack of specific investigation on the population ecology, it is not clear about its current status and survival potential; as well as the question whether the population's declining can lead to the differentiation of the local populations. In light of the superiority of Major Histocompatibility Complex (MHC) in population and conservation genetics,58 roughskin sculpin samples of the populations in Weihai and Dongying were used to explore the gene polymorphism of MHC Iα2. Combining with morphological comparison, the diversity of different populations from the macroscopic and microscopic perspective was also discusseed. Recently, the technique of cultivating Roughskin sculpin is becoming mature, and its breeding prospect and economic value is tremendous. While the research on fish immunology hasn't been reported so far. MHC, a kind of important immune factor that reacts to the protein antigens exists widely in vertebrates. In this study, MHC I genetic sequences were obtained by the RACE technology, and the MHC I and IGC1 recombinant proteins by constructing pET-3Oa expression vectors laid the basis for the study of MHC immune mechanism of Roughskin sculpin.After the T-test of 14 measurements and 1 I ratio traits of the samples, the result showed that there was no difference between male and female from the external feature and they could be used as one group for comparison.93 samples from two populations were used to study the I 1 ratio traits in the ways of composition analysis, cluster analysis, discriminant analysis, one-way ANOVA. and calculation of coefficient differences (C.D), it was found that only the discriminant analysis could set the two populations apart which reach the accuracy of 97.8%. The one-way ANOVA showed that the different Iraits of the two populations were mainly in the head (of the six different traits. three were in the head). This coeffieient of differences also showed that only two traits were close to subspecies level, which mean that the two populations have their tendency towards subspecies. Therefore, the differences between them still belong to their respective geographical populations.In this experiment, DNA from 58 samples in two populations of Weihai and Dongying were extracted,17 alleles were obtained from more than 300 cloned sequences. Among them, five alleles were in two individuals, suggested that there were at least three loci in MHC I a2. By analying MHC I a2 sequence, it was clear that of the existing 20 variation loci, the variation rate of amino acid was 16.9%. Besides, the amino acids variation rate in PBR region, which was higher than that of non-PBR region 12.3%(7/57), reached 50%(4/8). From the test of neutral selection, a conclusion could be drawn that the values of Tajima's D and Fs were both above 0 while the P values were above 0.01. It was estimated that a bottleneck effect or balance selection might happen. By MEGA3.1 the mean genetic distance of nucleotide sequences in Weihai population and Dongying population was 0.027 and of the amino acid sequences in the two populations was 0.047. By AMOVA (Analysis of Molecular Variance), the genetic variation, up to 90%, located within population, while 10% between populations. The value of Fst and (?)st was 0.11606 and 0.10521 respectively. Each P value was less than 0.01. This indicated that the two populations had moderate genetic differentiation.The MHC I gene, obtained in the experiment, was classic MHC I A which consisted of signal peptide, ectodomain, transmembrane region and cytomere. According to Gamle-Robson and Chou-Fasman's forecast of the secondary structure of MHC I A's protein molecules, the result showed that MHC I A protein belonged to mixed style, and by using the Swiss-model, the spatial structure of MHC I A's protein molecules of Roughskin sculpin was similar to the chicken.By constructing a pET-30a expression vector, the high expression value of MHC 1 A and IGC1 protein could be obtained. Through SDS-PAGH, it was detected thai two proteins existed in the sediments in the form of inclusion body. After being purified by High-Affinity Ni-IDA Resin affinity column, a pure protein was obtained, which would provide materials for monoclonal antibodies and MHC telramers in the future. |
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