| Many researchers have proved that Sika deer antler production was affected by many factors. In the present, the hot spot in Sika Deer Farming was to get high-yield by improving the unfavorable factors. If we could understand regulation mechanism of antler growth and development, antler production could be promoted by the development of new technologies.The basic means to improve sika deer antler production is to develop a method that is easy to operate, by which the results of selective breeding is reliable and the new Sika Deer would be high-yield antler production. Based on the backwardness deer selective breeding, for establishment the marker-assisted selection method, two sika deer populations of the Hubei BaiLuChun Industrial Corporation (n=302) and Jilin DongFeng farms(n=234)were selected to search single nucleotide polymorphisms (SNP) of Growth hormone (GH), melatonin receptor a (MTNR1A) and androgen receptor (AR) and analyze the correlation analysis between the SNPs in candidate genes and antler production traits. Meanwhile, protein structural and structural analysis of melatonin receptor gene was predicted by the bioinformatics software; the structure of MTNR protein can also be analyzed for confirming the functional units or domain and providing a target for genetic manipulation.The main results were showed as follows:1. SNPs screeningof GH, MTNR1A, and AR genes and their association with antler production of sika deer.(1)Polymorphism of GH gene and its association with antler production of sika deer: There are three mutations were found, including A176-G176(accession numbers: GQ438251.1), A45-G45, C139-T139(accession number:GQ438253.1) in growth hormone (GH) of two sika deer populations. The mutation A176-G176can lead to the change of restriction endonuclease recognition site Stuâ… , but the results of genotypes using PCR-RFLP analysis and correlation analysis between the gene polymorphism and the antler estimation value showed that there was no significant difference in two sika deer populations of the Five-three and Dongfeng farm (P>0.05); The mutation A45-G45and cannot lead to the change of restriction endonuclease recognition site, and there was no significant difference in two sika deer populations in the Five-three and Dongfeng farms (P>0.05); The mutation C139-T139cannot lead to the change of restriction endonuclease recognition site, and there was no difference in the sika deer population in the Five-three. But in Dongfeng farm, the antler production of sike deer with CT and CC genotypes were more than that with TT genotype (P<0.05).(2) Polymorphism of MTNR1A gene and its association with antler production of sika deer:There are three mutations of the MTNR1A genes exon2were found:c518-T518, G629-C629and C635-T635(accession numbers:JN038179) in these two sika deer populations. The mutation of C518-T518can change the restriction sites Ecol881, the correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value demonstrated that there was no significant difference in these two sika deer populations (P>0.05); the mutation of G629-C629can change the restriction endonuclease sites Mval, in the Five-three Farm, the correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value showed that the antler production of sika deer with CC genotype was more than that with GG genotype, and the difference was significant (P<0.05), and GC genotype was more than GC genotype, and the difference was significant (P<0.05); GC genotype was more than GG genotype, but the difference was not significant (P>0.05). The mutation of C635-T635can change the restriction sites. The correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value demonstrated that there was no significant difference in these two sika deer populations of the Five-three and Dongfeng farms (P>0.05).(3) Polymorphism of AR gene and its association with antler production of sika deer: There are two mutations of the androgen receptor gene (AR) were found:C75-T75in exon3and C256-T256in exon8(accession number:JF719040) in the two sika deer populations. The mutation of C75-T75can change the restriction endonuclease sites Rsaâ… In the Five-three Farm, the correlation analysis between Gene polymorphisms using PCR-RFLP method for genotyping and the antler production estimate value showed the antler production of sika deer with CT genotype was significant more than that with CC genotype (P<0.05); TC genotype was more than TT genotype (P>0.05), and TT genotype was more than CC genotype (P>0.05). In the Dongfeng Farm, TC genotype was significant more than CC genotype (P<0.05), TC genotype was more than TT genotype (P>0.05), and TT genotype was more than CC genotype (P>0.05). Correlation analysis between the gene polymorphism C256-T256mutation genotyping by SSCP-PAGE and the antler production showed that the polymorphism site was only found in the sika deer populations of Five-three Farm. And there was no significant difference between CC genotype and the TT genotype in Shuangyang sika deer antler production (P>0.05).2. Bioinformatic analysis of androgen receptor and melatonin receptor gene in sika deer(1) MTNR1A amino acid sequences of nine kinds of other vertebrates were analyzed using ClustalW multiple sequence model. The results showed that the amino acid of MTNR1A in sika deer was highly conserved sequence, just like other animals. The mutation of864G-C could change the animo acid. The predicted results of the physical and chemical characteristics of MTNR1A demonstrated that the change of animo acid could not change the stable feature of MTNR1A protein.(2) Phylogenetic analysis showed that MTNR1A gene in sika deer was consistency with the evolution of other species. The divergence time of sika deer which is ruminants as cattle is later than the higher animals before the birds. This study demonstrated that the sika deer follow the law of evolution of species during the course of evolution.(3) The results of protein prediction by SMART software found that there are seven transmembrane regionin MTNR1A protein sequence. The mutation of R288S was located in the864bp sites of conserved region of the NRF. Based on neural network algorithms and Hidden Markov models, we found two possible signal peptides,33-34and61-62, respectively.(4) Secondary structure features of MTNR1A protein showed that the changed protein by the864bp mutation was located between the sixth and seven transmembrane domain, the LOOP areas which was extracellular signaling molecule binding sites area. The region was exposed to the extracellular region and may be involved in downstream signaling pathway.(5) MTNR1A belongs to members of the GPCR family A. The results of homology modeling found that MTNR1A has55%homology with rhodopsin. The mutation of amino acids located in the extracellular loop ring position, which may be closely related with the combination of melatonin ligands and receptor. These results indicated that the melatonin receptor of the sika deer may be species-specific and special activation mechanism, which suggested that this locus could be the active site.(6) The results of IPA protein interaction network analysis showed that the melatonin receptor could interplay with many genes, transcription factors, adapter proteins, and form a complex regulatory network. MTNR1A is an important receptor of GPCR family, which could affect many physiological functions of the organism. The interaction network demonstrated that the heterodimers including MTNR1A and MTNR1B could perform its function together. Furthermore, MTNR1A could interact with CREB, ELK1/2and others to direct cell proliferation and differentiation. |
