Study On Genes Related To Ovine Prolificacy And Year-round Estrus

The melatonin receptor 1b(MTNR1B), inhibinβA(INHBA),kisspeptin, G protein-coupled receptor 54(GPR54),bone morphogenetic protein 6 (BMP6) were studied as candidate genes for the prolificacy and year-round estrus in sheep in the present study by techniques of PCR single-strand conformational polymorphism (PCR-SSCP),RT-PCR, gene cloning. The results were as follows:To analyze the structure and function of the melatonin 1b receptor (MT2) in sheep, single nucleotide polymorphisms were detected in exon 2 of sheep MT2 gene using genomic DNA from five sheep breeds by five primers. Polymorphisms were found.33 nucleotide mutations were revealed by comparing the mutant types with the wild types. Among them,14 give rise to deduced amino acid changes.However, none is likely to be associated with non-seasonal or seasonal estrus in sheep breeds tested. Sequence of exon 2 of MT2 of Small Tail Han sheep shows much closer phylogenetic relationship with predicted bovine and porcine MT2 than with human and mouse. The deduced amino acid sequence shows higher identity with the MT2 of cattle (95%) and pig (79%) than with human (76%) and mouse (71%).A rather high identity (61-63%) with the MT1 of sheep, human and mouse was also found. Compared with the other known MT2,35 unique altered amino acids were revealed. Albeit it also contains a NAXXY motif in transmembrane 7, both a DRY motif and a CYVCR motif were detected just downstream from its third transmembrane domain rather than NRY and CYICH found in other melatonin receptor groups.We presumed that it is possible that the structural changes make its binding function to the ligands attenuated or disrupted, and other genes (most probably MT1)were substituted in the progress of evolution, which ultimately resulted in no detectable expression in current breeds of sheep.Inhibins participate in the regulation of pituitary follicle-stimulating hormone synthesis and secretion, follicular maturation and steroidogenesis in the female. Inhibin (3a gene (INHBA)was studied as a candidate gene for the prolificacy of sheep. Single nucleotide polymorphisms of entire coding region and partial 3' untranslated region of INHBA were detected by PCR-SSCP in both two high fecundity breeds (Small Tail Han and Hu sheep) and six low fecundity breeds (Dorset, Texel, German Mutton Merino, South African Mutton Merino, Chinese Merino and Corriedale sheep).Only the PCR products amplified by primers 3,4 and 5 displayed polymorphisms.For primer 3,genotype CC was only detected in Chinese Merino sheep, genotype AA was detected in other seven sheep breeds. Genotype BB was only detected in Hu sheep. Only Hu sheep displayed polymorphism. Eight or four nucleotide mutations were revealed between BB or CC and AA, respectively, and these mutations did not result in any amino acid change. For primer 4, genotypes EE, EG and GG were detected in Dorset and German Mutton Merino sheep, genotypes EE, EF and FF were detected in Chinese Merino sheep, only genotype EE was detected in other five sheep breeds. Only Dorset, German Mutton Merino and Chinese Merino sheep displayed polymorphism. Sequencing revealed one nucleotide mutation (114G→A) of exon 2 of INHBA gene between genotype FF and genotype EE, and this mutation did not cause any amino acid change. Another nucleotide change(143C→T) was identified between genotype GG and genotype EE, and this mutation resulted in an amino acid change of serine→leucine.For primer 5,genotypes KK and KL were detected in German Mutton Merino and Corriedale sheep, genotypes KK, LL and KL were detected in other six sheep breeds. Genotype MM was only detected in Hu sheep.All of these eight sheep breeds displayed polymorphism. Sequencing revealed one nucleotide mutation (218A→G) of exon 2 of INHBA gene between genotype LL and genotype KK, and nine nucleotide mutations between genotype MM and genotype KK. These mutations did not alter amino acid sequence.The partial sequence (395 bp for exon 1 and 933 bp for exon 2) of INHBA gene in Small Tail Han sheep (with genotype KK for primer 5)was submitted into GenBank (accession number EF192431).Small Tail Han sheep displayed polymorphisms only in fragment amplified by primer 5.The Small Tail Han ewes with genotype LL had 0.53 (p<0.05)or 0.63 (p<0.05)lambs more than those with genotype KL or KK, respectively. The Small Tail Han ewes with genotype KL had 0.10 (p>0.05) lambs more than those with genotype KK.According to the sequence of the predicted Bos taurus KiSS-1 gene and its receptor GPR54 gene, four pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 of KiSS-1 gene and exon 1,exon 2 and partial exon 5 of GPR54 gene in high fecundity and year-round estrous breeds(Small Tail Han and Hu sheep) and low fecundity and seasonal estrous breeds (Dorset, Texel and Corriedale sheep) by PCR-SSCP. Polymorphisms in exon 1 of KiSS-1 gene were found in year-round estrous Small Tail Han sheep (AA,BB and AB genotypes) and Hu sheep (AA and CC genotypes), no polymorphism was found in seasonal estrous sheep breeds (only AA genotype).Polymorphisms in exon 2 of GPR54 gene were found in year-round estrous Hu sheep (DD and EE genotypes), no polymorphism was found in year-round estrous Small Tail Han sheep and seasonal estrous sheep breeds (only DD genotype).No polymorphism was detected in exon 1 and partial exon 5 of GPR54 gene in five sheep breeds. The polymorphic genotypes were sequenced. While compared the BB genotype with the AA genotype, one nucleotide mutation (G73A) was detected, which resulted in amino acid change, Val25Met. Seven nucleotide mutations were detected from AA to CC genotype (C19T, C33T, T34C,C63A, T64C,C72G, C77T),and among them 4 make amino acid changes, that is, Arg7Trp,Phel2Leu, Asn24Lys, Ala26Val.While compared the EE genotype with the DD genotype, two nucleotide mutations (T269C, A320C) were detected, which give rise to amino acid changes, Met90Thr and Asp107Ala, respectively. Genotype frequencies of AA,BB and AB were 0.62,0.05 and 0.33,respectively. The Small Tail Han ewes with genotypes BB and AB had 0.78 (P<0.05) or 0.45 (P<0.05) lambs more than those with genotype AA;the Small Tail Han ewes with genotype BB had 0.33 (P>0.05)lambs more than those with genotype AB. These results preliminarily indicate that the KiSS-1 gene may have some association with prolificacy and year-round estrus in sheep.The bone morphogenetic protein 6 gene was studied as a candidate gene for the prolificacy of Small Tail Han sheep.Single nucleotide polymorphisms(SNPs) of exons 5,6,7 of BMP6 gene were detected in high fecundity breeds (Small Tail Han and Hu sheep) and low fecundity breeds (Dorset, Texel and Corriedale sheep) with three,pairs of primers by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP).No polymorphisms were detected in the amplified regions of the three pairs of primers in the five sheep breeds tested, which may means that the sequences of exons 5,6,7 of BMP6 gene were rather conserved, and these regions may not be the functional domains that affect the prolific performance of sheep.On the other hand, partial sequences of BMP6 mRNA were cloned from sheep and goat,695bp and 699bp respectively, and the sequences of intron between exon 3 and exon 4 with some of the two flanking exons were also sequenced. The two BMP6 sequences show high identical with 99.28% in nucleotide and 98.1% in amino acid sequence, and besides an insert of alanine in goat, only 4 alterations of amino acids were found between them (211 in total).Compared the ovine nucleotide and amino acid sequence of BMP6 with that of cattle, human, mouse and rat, identity of 97.04%,81.82%,84.68%,85.06% and identity of 99.05%, 91.43%,90.48%,92.38% were revealed respectively.The results suggested that although the change of nucleotides is a little big, but the sequence of amino acid of BMP6 is very conservative among species.