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Authors: D. DIVYA
Language: en
Type: Thesis
Pages: 241
Series/Report no.: D;539
Abstract: The present study was aimed at evaluating random bred broiler and layer lines maintained at Directorate of Poultry Research, Rajendranagar, Hyderabad with respect to expression profiling, epigenetic and functionality of promoter of BMP3 and BMP4 genes. BMP3 acts as a negative regulator for osteogenesis and as a modulator of osteogenic activity of other BMPs in vivo while BMP4 shows osteogenetic activity in vivo and in vitro. The functionality of promoters was elucidated by means of reporter GFP marker in vivo. BMP3 gene promoter was divided into two fragments of 501 and 786 bp while that of BMP4 was divided into fragments of 541 bp and 1.1 kb size. Larger fragments were better promoters. Both the promoters displayed polymorphism by means of single strand conformation polymorphism study using blood samples of broiler (286) and layer (270) birds. For BMP3 gene promoter, haplotype, h1 was predominant (0.735 and 0.62) and h3 was the least frequent (0.046 and 0.04) in broiler and layer lines, respectively. In case of BMP4, h1 haplotype had higher (0.90 and 0.97) and h2 had lower (0.03 and 0.01) frequencies in broiler and layer lines, respectively. Both the populations were in Hardy-Weinberg equilibrium. A non-significant effect of haplogroup was observed on growth traits in both the lines for both the genes with the exception of BMP4 gene on body weight at 35 and 42 days of age and growth rate between day 28 and day 42 and that of BMP3 gene on body weight at day 14 and growth rate between day 1 to 14 and day 14 to 28 of age in broilers only. Expression profiling during pre- and post-hatch periods was explored using real time PCR. The BMP3 gene expression was the highest and lowest at embryonic day (ED) 9 and 5 in broilers and on ED7 and ED8 in layers, respectively. During post-hatch period, the highest and lowest expressions were observed on day 42 and 14 in broiler and 1 and 28 in layer line, respectively. The BMP4 gene expression level during pre-hatch period was the highest and lowest on ED8 and ED14 in broilers and ED17 and ED10 in layers while during post hatch, it was on day 42 and 28 in broiler line and on day 1 and 28 in layers, respectively. The fold change in expression was also calculated by taking expression values on ED5, ED18 and day 1 as calibrators against which the expressions at different days were compared. When the expression values of BMP4 gene of broiler and layer chickens were compared during pre-hatch and post-hatch period, a significant effect at different periods of growth was observed. The complete open reading frame of both genes was characterized through real time PCR, SDS-PAGE and Western blot where many mutations were observed. Protein structure was predicted at different (primary, secondary and tertiary) levels via bioinformatics tools. The nucleotide and protein sequences were compared with other avian and non-avian species via phylogeny. Significant effect (P≤0.05) of BMP3 gene expression on breast angle and giblet percentage in broiler line and on leg and breast muscle percentage in layer line and that of BMP4 gene expression on body weight at day 28 in layer line and on shank length and giblet percentage in broiler line was observed. Further, DNA methylation, a heritable epigenetic marker was executed for BMP3 and BMP4 gene promoter on day 1 and day 42 post-hatch in broiler and layer lines selected based on high and low expression of genes where a non-significant difference (P≤0.05) was observed with respect to the methylation percentages of CpG’s in both the genes together with a strong negative correlation between expression value and methylation level. The overall methylation per cent of BMP3 gene promoter was 5.67 ± 1.85 on day 42 and 3.82 ± 1.90 on day 1 in broiler line over layer population i.e. 5.77 ± 0.03 on D1 and 7.54 ± 1.87 on D42. While, with respect to BMP4 gene promoter, the overall methylation per cent in broiler and layer was 7.97 ± 2.01 and 10.23 ± 3.9 on D1 and 9.13 ± 2.67 and 15.91 ± 0.64 on D42, respectively. An increasing trend of methylation was observed with age of bird. Finally, it can be concluded that the BMP3 expression was higher in layer line than in broiler line while, BMP4 expression was comparatively higher in broiler than layer line with higher methylation percentage observed on day 42 over day 1 in both the lines for both the genes. The larger fragment of promoter region in both the genes acted as better promoter for regulating gene expression under in vitro cell culture system.
Subject: Animal Genetics and Breeding
These Type: Ph.D
Issue Date: 2017-06-08
Appears in Collections:Thesis

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