Manzoor Sidra, Nadeem Asif, Maryam Javed, Hashmi Abu Saeed, Imran Muhammad, Babar Masroor Ellahi
Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, 54000, Pakistan.
Virtual University of Pakistan, Lahore, 54000, Pakistan.
Trop Anim Health Prod. 2018 Feb;50(2):275-281. doi: 10.1007/s11250-017-1426-1. Epub 2017 Sep 30.
Osteopontin gene is regarded as a plausible candidate in mammary gland differentiation and development, expressed by variety of cells, tissues, and biological fluids including milk. The current study was performed in two phases. In the first phase, Osteopontin gene polymorphisms were identified and associated with milk composition such as ash, milk fat, SNF, lactose, and protein. In the second phase, milk samples from five healthy mastitis-free Nili Ravi buffaloes were analyzed for expression of Osteopontin gene at transition (day 15), mid (day 90), and end (day 250) stage of their second lactation. Briefly, blood samples were collected from Nili Ravi buffalo to isolate the genomic DNA, specific primers were designed for PCR amplification. The amplified PCR products were sequenced bi-directionally. Six polymorphisms were identified in the coding region and four in the intronic region of the gene. The results showed that SNP g.38329758 T > C causing substitution of valine to alanine (V127A) was associated with high milk protein. For mRNA expression analysis, somatic cells were separated from milk samples for RNA isolation. Analysis of differential gene expression data has permitted us to illustrate the expression pattern of osteopontin gene in lactating buffalo. The Osteopontin gene was found to be transcribed among all three lactation stages, but expression was observed with the highest value (fold change) in peak lactation and remained elevated till the end of lactation. Identified gene marker may be helpful for the prediction of superior animal for selection. The presented study also gave an insight into the genetic screening and lactation biology of riverine buffalo, offering direction for future research in lactating buffalo.
骨桥蛋白基因被认为是乳腺分化和发育中一个可能的候选基因，它由包括乳汁在内的多种细胞、组织和生物体液表达。本研究分两个阶段进行。在第一阶段，鉴定了骨桥蛋白基因多态性，并将其与乳成分如灰分、乳脂肪、非脂乳固体、乳糖和蛋白质相关联。在第二阶段，分析了5头健康无乳腺炎的尼里-拉菲水牛在第二个泌乳期的过渡阶段（第15天）、中期（第90天）和末期（第250天）的乳汁样本中骨桥蛋白基因的表达情况。简要来说，从尼里-拉菲水牛采集血液样本以分离基因组DNA，设计特异性引物用于PCR扩增。对扩增的PCR产物进行双向测序。在该基因的编码区鉴定出6个多态性，在内含子区鉴定出4个多态性。结果表明，导致缬氨酸替换为丙氨酸（V127A）的SNP g.38329758 T>C与高乳蛋白相关。对于mRNA表达分析，从乳汁样本中分离体细胞以进行RNA分离。差异基因表达数据分析使我们能够阐明骨桥蛋白基因在泌乳水牛中的表达模式。发现骨桥蛋白基因在所有三个泌乳阶段均有转录，但在泌乳高峰期表达值（倍数变化）最高，并且一直持续升高直至泌乳结束。鉴定出的基因标记可能有助于预测用于选择的优良动物。本研究还深入了解了河流水牛的遗传筛选和泌乳生物学，为泌乳水牛的未来研究提供了方向。
