| The Eastern Zhejiang White goose (EZWG), which is known for its rapid growth rate, superexcellent meat quality, early sex mature and disease resistance, classified to the medium body size of Chinese white goose.It has been developing speedily in animal science construction change in the latter years.Meanwhile, the female EZWG reproduction ability is low because of strong broodiness, unable to adapt to modern industrial production. Therefore, the objective of this study was to determine molecular mechanism of EZWG broody behavior and to know how genetic factors to induce it, moreover, to know the genetic laws of its economic traits in order to develop molecular-assisted selection using in goose breed. Prolactin is a polypeptide hormone secreted by the anterior pituitary gland and affects a variety of physiological functions in birds,such as reproduction, growth and development, osmoregulation, metabolism, behavior and immunoregulation. The onset of broodiness in birds was initiated by an increase of prolactin level in plasma that was transported into the brain via the choroid plexus to induce the behavior. Thus, prolactin gene, prolactin receptor gene and pituitary specific transcription factorl (which is one of prolactin expression affected factor) were selected as candidate gene to study the molecular mechanism of EZWG broodiness. At first, the three candidate genes DNA sequences were obtained by PCR using EZWG genomic DNA as template.Then, the mRNA expression of prolactin and prolactin receptor in the hypothalamus, pituitary gland and ovary of adult female geese at egg-laying, out-of-lay and incubating stages were carried out by real time PCR using SYBR Green I. At last, the SNPs of prolactin, prolactin receptor and pituitary specific transcription factorl in EZWG were detected by PCR-SSCP. The associations between these SNPs polymorphisms and some goose economic traits were analyzed by using the SPSS 13.0 general linear model (GLM). Results were obtained as follows:A sequence of 5917 bp in length of EZWG complete prolactin cds was obtained by PCR amplification and sequencing, which has five exons (21…48,1557…1738, 2135…2242,3631…3810,5706…5897) and four introns, and it was submitted to Genebank, accession number DQ660983.A sequence of 1305 bp in length of EZWG partial exon of prolactin receptor gene was obtained by PCR amplification and sequencing, its Genebank accession number was DQ660982.Five sequences containing five exons of pituitary specific transcription factor 1 of EZWG were obtained by PCR amplification and sequencing.They were 963 bp, 803 bp,253 bp,169 bp and 210 bp in length respectively and its Genebank accession numbers were EF522106, EF457938 (803 bp and 253 bp) and EF413635.A sequence of 235 bp in length of EZWG Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) was obtained by PCR amplification and sequencing. It contains two exons (1…68,176…235) and an intron (69…175) and its GeneBank accession number was DQ454070.Meanwhile, a sequence of 209 bp in length of EZWG GAPDH mRNA was obtained and its GeneBank accession number was DQ821717.Based on normalization with GAPDH gene, prolactin and prolactin receptor mRNA expression of EZWG had significant difference (P<0.05)at egg-laying, out-of-lay and incubating stages by real time PCR. Prolactin mRNA in the hypothalamus and pituitary gland of incubating geese was significantly higher (P<0.05)than that of out-of-lay. Meanwhile, prolactin mRNA in the ovary of incubating EZWG was very significantly higher (P<0.01)than that of out-of-lay, whereas no any difference was observed between egg-laying and out-of-lay or brooding (P>0.05). Prolactin receptor mRNA of incubating EZWG was significantly higher (P=0.001)than that of out-of-lay. And the results also indicated that the prolactin and prolactin receptor mRNA had its highest levels in pituitary gland, then followed hypothalamus and the lowest in ovary at each stage.The correlation between the prolactin and prolactin receptor expressions of EZWG was highly significant(P<0.01)(Sig. Two-tailed), and Pearson correlation coefficient was 0.698.Fourteen SNPs were identified in EZWG prolactin gene by PCR-SSCP, which included thirteen of introns and one of exon. The intron SNPs were at 479 bp (C-A), (G-A),5070 bp (T-C),5170 bp (G-C),5343 bp (T-C),5344 bp (G-A),5478 bp (T-G),5612 bp (G-T) and exon SNPs was at 5856 bp (A-C).All of these SNPs were synonymous substitution that did not led to coding amino acid change.Two SNPs in exon of prolactin receptor gene were identified in EZWG by allele specific PCR and were at 694 bp (A-C) and 981 bp (A-G).The mutation at 694 bp (A-C) was synonymous substitution, coding Histidine, at 981 bp (A-G) was non-synonymous substitution, leading to amino acid change from Asparagine to Glycine.The two SNPs of prolactin receptor gene related to geese broodiness by detection of different geese with different broody levels of China, combining with the analysis of PRL gene balance of Hardy-weiberg. Therefore, PRL and PRLR genes have major effect on broodiness of EZWG.The genetic effect of the prolactin and prolactin receptor SNPs on the growth and reproduction traits was analyzed by the GLM procedure of SPSS software, the results showed that the highly polymorphism P-1 locus of prolactin gene has significant effect (p<0.05) on EZWG weight and P-8 locus of prolactin gene has significant effect (p<0.05)on EZWG chest broad.The SNPs locus at 981 bp (A-G), loci of P-1 and P-8 of prolactin have significant effect (p<0.05)on progeny birth weight of EZWG. |
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