Cloning And Studies On Expression Characteristics And Genetic Effects Of Goose PIT-1 Gene

Pituitary-specific factor 1 (Pit-1) was encoded by PIT-1 gene, it is a transcription factor expressed in cephalic of Pituitary gland with essential function. PIT-1 regulates the transcription of PRL, GH, TSH-βand itself by binding to the promoter of them, and plays important roles in the growth, development, reproduction and immunity by regulates the expression of these genes.In this study, the cDNA sequence of PIT-1 gene was first cloned in Shitou goose, Wanxi white goose and Zi goose, and the bioinformatics analysis on the CDS and amino acid sequences was conducted; then analysis of genetic variation of PIT-1 was carried out in Shitou goose, Wanxi white goose, Zi goose, Zhedong white goose, Siji goose and landosie goose, the genotypes distribution in different populations was compared and the effects of genotypes on early bodyweight and carcass traits was analyzed; further, gene expression of PIT-1 gene, PRL gene and GH gene in different developmental stages in Wanxi white goose was investigated by Real-time PCR technology to explore the expression characteristics. The main results were as follows:1. Two cDNA sequences of 1014 bp size and the sequences of intron 2 and intron 2a was gained by the methods of RT - PCR and sequencing in Wanxi white goose, Shitou goose and Zi goose; the CDS of PIT-1 gene was 1011 bp in length, and the identity with human, mouse, turkey, chicken and duck were 63.2%, 68.2%, 83.5%, 84.3% and 97.2%, respectively; the size of intron 2 was 819 bp, and intron 2a was 333 bp, their identity with duck introns were 83% and 89.2%; the bases in position 485 and 746 was different in two cDNA sequences.2. Bioinformatic analysis was conducted on CDS sequences, the differences in nucleotides changed the amino acids at position 162 and 250, there were high identity between the amino acids sequence with turkey, chicken and duck. There were 15 phosphorlated sites in amino acids sequence, most of the amino acids were hydrophilic, the there was no transmenbrane helices in the sequence. The second structure of amino acids was consisted of alpha-helix (154AA), extended strand (16AA) and random coil (166AA). The structure of amino acids was characterized with POU specific conserved region, and make sure that this is the sequence of Pit-1.3. Two insertion/deletion were detected in intron 1 of goose PIT-1 gene, the distribution of this variation was significantly different in different populations, and the landosie goose showed as monomorphsim. Statistic analysis showed that, this variation was remarkable correlated with the early bodyweight of Wanxi white goose, allele B ,which has the inserted fragments was benefit to the gain of early bodyweight; at the same time, this variation significantly affected the heart ratio of Shitou goose, AB was higher than AA. This conclusion indicated that, the variation may affect different traits in different type of populations. Three SNPs of PIT-1 gene were detected in 6 populations, among them the SNP in intron and of position 364 were only appeared in landosie goose; genotype distribution was significant between populations, and EE genotype was detected only in landosie goose. Statistic analysis showed that the early bodyweight of DD genotype in Shitou and Zi goose was significantly higher than CD, and bodyweight of CD genotype in Zhedong white goose was significant than that of CC, which suggested that allele D was positive for bodyweight; ratio of abdominal fat of DD genotype was remarkable higher than CD in Shitou goose, however the difference of carcass ratio was not significant between DD and CD genotype in Zi goose, and CC was significantly higher than CD, which may indicated that, the effects of genotypes on traits depend on the commercial type of populations. Landosie goose shown extreme remarkable differences with other 5 populations on the variations, it shown monomorphism in both variations, which may due to the different originations or related to the long time breeding for specific productions; the AA genotype of insertion/deletion variation and the EE genotype of SNPs variation may be more beneficial to Foie Gras trait, and could be used as the assisted molecular marker for breeding.4. Ontogeny expression of PIT-1 gene, GH gene and PRL gene was conducted by Real-time PCR in Wanxi white goose, the results showed that, expression of PIT-1 gene and GH gene in pituitary of male shown differences with that of female during 0 - 4 weeks'age, and the peak and valley of GH gene expression in hypothalamus appeared at different time of male and female, which suggested sexual dimorphism of gene expression during 0 - 4week, and different effects on traits in male and female. Within the sex, GH gene expression patterns of male in Pituitary and hypothalamus was identical, and differences existed in two tissues in female at 0 - 2 and 30 week's age, which suggested that there could be some differences in the mechanism of regulation on GH gene in male and female tissues. The expression patterns of PRL gene shown differences between tissues during 2 - 10 weeks, changes in pituitary and hypothalamus was the same, and before 15 weeks, changes in ovary was opposite with hypothalamus, this may due to the regulation of PRL gene by different factors in different tissues. Expression characteristics of PIT-1, GH and PRL in Pituitary were also compared in this study, and we found that, the expression pattern of GH gene was the same as PIT-1 gene in both sexes, which notifies that the expression of GH gene changed in response to PIT-1 gene, and it was under the genetic regulation of PIT-1. However, the pattern of PRL was not the same as PIT-1 at all stages, this result was similar to previous study, and it could be concluded that the expression of PRL may under the combined effects of PIT-1 and other factors.