| Melanopsin is newly found opsin with seven transmembrane domain, and it belongs to G protein-coupled receptor (GPCR). Melanopsin has light sensitivity, similar effect with Vitamin A, and function of signal transduction. Melanopsin is encoded by Opn4gene and expressed exclusively in intrinsically photosensitive retinal ganglion cells (ipGCs) of retina. Medicated by Melanopsin, ipGCs can feel light signal and cas it onto suprachiasmatic nucleus. Therefore, ipGCs has fuctions of non vision imaging system such as pupillary light reflex, neuroendocrine regulation, light signal transmission l3]. The research on Melanopsin recently has changed people’s cognition on retinal photoreception, and it makes ipGCs to be the third kind of photosensory cell after cone cell and rod cell.Swine is a large-scale livestock with poor vision and special habit of sleepiness, perceiving the environment mainly by its well-developed sense of smell. Light plays an essential role in the adjustment of the biological rhythm, making it an interest to research on Melanopsin as a photoreceptor and circadian rhythm regulator in swine. To reveal the swine Opn4gene express distribution structure, tissue expression distribution, and study its expression differences between day and night in swine retinal and expression in different stages of the growth regularity and Opn4gene polymorphism. The swine opn4gene was to clone for structure and function analyzing in this experiment, afterwards, the study on the gene mRNA expression pattern in different time of the day and different growing stage of the swine as well as combining the PCR-RFLP and DNA sequencing method to figure out Opn4gene polymorphism were also conducted. The purpose of the experiment is to reveal the swine Opn4gene function and regulation mechanism of the biological rhythms, and then help to understand the relationship between visual and optical signal changing and corresponding respondence of animals action. Results and conclusion from this study are as follows:1. The swine Opn4gene sequencing and bioinformatics analysisA1437bp coding region of Opn4gene was amplified by PCR technique from the genome of swine, and further analysis showed the relative opsin contained478amino acids. It has seven transmembrane regions, and the main secondary structure is helix and there is1structure domain.It has a high homology compared to human, emerging a similarity of85.73%and81.38%at the amino acid level. Additionally,3highly-conserved functional domains Pfam were forecasted in both human and swine. In human, the gene has the same motif location but different amino acid sequence with swine for Ser(S) at157, Tyr(Y) at345while Thr(T) and His(H) respectively in swine. The Opn4gene showed a highest homolog with bovine through Phylogenetic relationships demonstrating a silimilar regulation pattern of photoreceptor and circadian rhythm between swine and bovine which is maybe a result of the artificially domestication.2. Analysis of tissue-specific expression profile of the swine Opn4geneTissue distribution was analyzed by the sequencing and the RT-PCR in13different tissues including Heart, liver, spleen, lung, kidney, left brain, right brain, cerebellum, fat, muscle, hypothalamus, retina and brain stem. The Opn4gene was defected in3different tissues,which was retina, hypothalamus and brain stem, respectively. Because no research ever done showed that Opn4gene expressed in the brain stems, the repeating tests in the experiments was carried out to prove the discovery, which the Opn4gene can express a small amount in the brain stem, is true,. However, what the specific role an Opn4gene plays in the brain stem cells needs further studies.3. The swine Opn4gene analysis of the mRNA expression level in different growth stages and day-night patternThe expression level of Opn4in the day was significantly higher than in the night (P<0.01). During the growing period, Opn4started expressing as early as34d in the embryonic phase with the lowest level (.P<0.01), and came to a peak1day after birth (P<0.01), and then stayed at a moderate standard. The difference of Opn4expression among the swines at1month,4months, and84months was not significantly different (P>0.05). Together, results showed that the Opn4gene can regulate the biological rhythm of the swines and play an important role in mediate the responding to the light for the postnatal swines.4. Analysis of genetic diversity of the swine Opn4geneIn this study, genetic variations of Opn4genes were detected by PCR-RFLP and DNA sequencing techniques in380individuals from two swine breeds (Tibetan swine160and import swine200). We analyzed the genetic variation of all the exons and intron9of Opn4in this study and found that11mutation sites were detected in intron1, intron9and exon4, exon5. exon7, exon8, and only the mutation site with exon7caused amino acid to be changed, which original Tyr mutated into termination codon TAG. The PCR-RFLP analysis showed that there were three genotypes:CC, CG and GG. Homozygosity and heterozygosity of this site in the two groups of Tibetan swine and import swine were similary and PIC values were both moderate polymorphic.The χ2test indicated that the genotypic frequencies of Opn4gene in the two populations do not meet Hardy-Weinberg equilibrium (P<0.01)... |
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