Sidra Manzoor (2010-VA-92)
Exploration Of Genetic Polymorphisms And Differential Expression Analysis Of Bovine Alpha-Lactalbumin And Osteopontin Genes Involved In Milk Composition - 2017. - 144p.;
Economically important traits of dairy animals are usually controlled by a large number of genes. The identification of the single nucleotide polymorphisms in potential genes has been associated with economically important traits. During lactation, mammary epithelial cells produced large amounts of specific milk proteins. Due to the expression sites, physiological properties and chromosomal localization, LALBA and SPP1 genes might be considered as candidate genes for milk composition in buffalo. Alpha-lactalbumin (LALBA) gene has been reported to be highly transcribed in transition and peak phase while late lactation exhibited its decline with progressive rise in SPP1 expression. This project was designed to investigate the effect of single nucleotide polymorphism that influencing the gene expression thus modulates the milk protein content in Nili Ravi. Samples of unrelated Nili-Ravi buffalo were collected from two Government, Buffalo Research Institute, Pattoki, and Livestock Production and Research Institute (LPRI) Bahadarnagar Okara, livestock farms. Milk samples were collected at 15, 90 and 250 days lactation for expression analysis. The genomic DNA was extracted by using the standard Phenol Chloroform Isoamyl alcohol (PCI) protocol. Specific set of primers was designed for the amplification of the LALBA and SPP1 genes. The amplified PCR products were sequenced for the identification of SNPs. To determine the differential expression of bovine LALBA and SPP1 genes, RNA was isolated from milk samples using the TRIzol reagent and converted it into cDNA. Taqman probes were used that are specifically designed to detect and target the DNA sequence. Five intronic polym orphic sites were identified in LALBA while exonic regions exhibited a complete homology with reference sequence. Additionally, eleven polymorphisms were identified in bovine SPP1 gene, six were in coding region and five were
Summary
122
found in intronic portion of the gene. The analysis and correlation of all identified polymorphism was done by using SNPs data analysis software “SNPator”. Results obtained from expression study was stored in in-build software of Real Time PCR and Cycle threshold (Ct) values of LALBA and SPP1 mRNA were compared in individuals of Nili-Ravi buffalo to determine the variation in expression levels. The LALBA gene expression was observed highest in transition phase with a gradual decrease of expression in mid and late lactation. The sample, NR-5, was observed highly expressed (79.30) while NR-2 with low expression (19.28) for alpha lactalbumin in early lactation. The change in LALBA regulation at same stage was considered due to genetic variation of the respective animal. While the SPP1 gene expression was observed with the highest values in peak lactation and remains elevated in late lactation. NR-4 has the highest (72.27) expression among all mastitis free healthy animals while NR-2 was observed with low expression. Thus, the identified SNPs might be used as genetic marker for milk production traits. Gene expression patterns may also help us to understand the molecular mechanisms of bovine LALBA and SPP1 genes influencing milk composition. However, the expression of both genes was considered in a correlation with other genes involved in milk production pathway. Also, the mutational effects of other milk proteins might be involved in determining the expression pattern of both genes in selected animals. Therefore, further studies are likely to explore the regulation of milk protein genes and their translational efficiency during the course of lactation in dairy animals.
Molecular Biology & Biotechnology
Phd. Thesis
2830-T
Exploration Of Genetic Polymorphisms And Differential Expression Analysis Of Bovine Alpha-Lactalbumin And Osteopontin Genes Involved In Milk Composition - 2017. - 144p.;
Economically important traits of dairy animals are usually controlled by a large number of genes. The identification of the single nucleotide polymorphisms in potential genes has been associated with economically important traits. During lactation, mammary epithelial cells produced large amounts of specific milk proteins. Due to the expression sites, physiological properties and chromosomal localization, LALBA and SPP1 genes might be considered as candidate genes for milk composition in buffalo. Alpha-lactalbumin (LALBA) gene has been reported to be highly transcribed in transition and peak phase while late lactation exhibited its decline with progressive rise in SPP1 expression. This project was designed to investigate the effect of single nucleotide polymorphism that influencing the gene expression thus modulates the milk protein content in Nili Ravi. Samples of unrelated Nili-Ravi buffalo were collected from two Government, Buffalo Research Institute, Pattoki, and Livestock Production and Research Institute (LPRI) Bahadarnagar Okara, livestock farms. Milk samples were collected at 15, 90 and 250 days lactation for expression analysis. The genomic DNA was extracted by using the standard Phenol Chloroform Isoamyl alcohol (PCI) protocol. Specific set of primers was designed for the amplification of the LALBA and SPP1 genes. The amplified PCR products were sequenced for the identification of SNPs. To determine the differential expression of bovine LALBA and SPP1 genes, RNA was isolated from milk samples using the TRIzol reagent and converted it into cDNA. Taqman probes were used that are specifically designed to detect and target the DNA sequence. Five intronic polym orphic sites were identified in LALBA while exonic regions exhibited a complete homology with reference sequence. Additionally, eleven polymorphisms were identified in bovine SPP1 gene, six were in coding region and five were
Summary
122
found in intronic portion of the gene. The analysis and correlation of all identified polymorphism was done by using SNPs data analysis software “SNPator”. Results obtained from expression study was stored in in-build software of Real Time PCR and Cycle threshold (Ct) values of LALBA and SPP1 mRNA were compared in individuals of Nili-Ravi buffalo to determine the variation in expression levels. The LALBA gene expression was observed highest in transition phase with a gradual decrease of expression in mid and late lactation. The sample, NR-5, was observed highly expressed (79.30) while NR-2 with low expression (19.28) for alpha lactalbumin in early lactation. The change in LALBA regulation at same stage was considered due to genetic variation of the respective animal. While the SPP1 gene expression was observed with the highest values in peak lactation and remains elevated in late lactation. NR-4 has the highest (72.27) expression among all mastitis free healthy animals while NR-2 was observed with low expression. Thus, the identified SNPs might be used as genetic marker for milk production traits. Gene expression patterns may also help us to understand the molecular mechanisms of bovine LALBA and SPP1 genes influencing milk composition. However, the expression of both genes was considered in a correlation with other genes involved in milk production pathway. Also, the mutational effects of other milk proteins might be involved in determining the expression pattern of both genes in selected animals. Therefore, further studies are likely to explore the regulation of milk protein genes and their translational efficiency during the course of lactation in dairy animals.
Molecular Biology & Biotechnology
Phd. Thesis
2830-T