The present study was undertaken in 60 Jaffarabadi buffaloes for molecular characterization of the coding regions of Interleukin 8 gene through PCR-RFLP technique and also to study its association with occurrence of mastitis. A set of primers were designed by using primer-3 software covering 853 bp nucleotides of IL-8 gene for Jaffarabadi buffalo. The PCR-RFLP was done for all the samples using Hae III and Dra I restriction enzymes. The amplified fragments of exon-3 in Interleukin -8 gene exhibited monomorphism.
India continued to be the largest milk producing nation with an anticipated milk production of 155.49 million tonnes during 2015-16; buffalo contribute 56% milk (NDDB, 2016). Jaffarabadi buffalo is on the major milch breeds in Gujarat with the native breeding of Saurashtra region especially in and around Gir forest viz., Junagadh, Bhavanagar, Jamnagar, Porbandar, Gir-Somnath, Amreli and Rajkot districts. Average lactation milk yield of Jaffarabadi buffalo is 2150-2340 Kg (Nivsarkar et al., 2000). The breed has been characterized for various genes at the molecular level like Bola-DRB3 gene (Acharya et al., 2002), OLR1 gene (Shabir et al., 2011) and leptin gene (Yadav et al., 2015). However, Interleukin-8 gene which is considered to be associated with the mastitis resistance has not been characterized in the breed. Interleukin-8 (IL-8) is a cytokine of chemokine family and is produced by both leukocyte cell and non – leukocyte cell types including lymphocytes, neutrophils, monocytes, macrophages and epithelial cells. IL-8 has many biological activities like, recruiting and activating neutrophils during mastitis, neutrophil degranulation, stimulating phagocytosis, recruiting T-lymphocytes and preventing retention of placenta in calved animals. Therefore, the present research has been undertaken with the objectives to characterize Interleukin-8 gene in Jaffarabadi buffaloes and to explore genetic polymorphism of Interleukin-8 (IL-8) in Jaffarabadi buffaloes.
Materials and Methods
A total of 60 Jaffarabadi buffaloes from cattle breeding farm, Junagadh Agricultural University, Junagadh were selected for blood sample collection. About10 ml blood was collected from jugular vein of each buffaloes in vacutainer. The DNA was isolated using phenol-chloroform extraction method with little modifications (Sambrook and Russell, 2000). A total of five μl DNA samples were used for spectrophotometry. Optical density (OD) values at 260 nm and 280 nm were measured using small volume UV spectrophotometer (Nanodrop). The DNA samples with an OD260: 280 ratio of 1.8 to 2.0 were further subjected to agarose gel electrophoresis for quality check. The qualities of the isolated DNA were checked on 0.8 % agarose gel. Total five primers (forward and reverse) were designed which covers entire IL-8 gene. The sequence was retrieved from the NCBI gene bank available online. The conserved regions were detected using MegAlign software (Table 1).
Table 1: List of Primers
|S. No.||Primer Sequence 5’-3’||Primer Length (bp)|
|Primer 1||Forward 5’-GGGCGGAGGTTGCGTATT-3’
|Primer 2||Forward5’-GACGAGCTTCAGGCAACTATCA-3’ Reverse 5’-ATATTAAATGCCATGGAGACAAA-3’||22
|Primer 3||Forward 5.-TGGAAGAATCCAGCAAAGTTC-3.
|Primer 4||Forward 5.-CCAATCGATCTGGAAATCCT-3.
|Primer 5||Forward 5.-CACAAACAGAAAGACCTCTTAGTCA-3.
A standard PCR Protocol for 25 μl reaction mix was prepared. The details of the PCR master mix is as per Table 2. The restriction enzymes Hae III and Dra I were used to digest the amplicons of IL8 gene. The PCR amplified products of IL8 gene were cleaved with the Hae III restriction enzyme in 30μl reaction volume at 37oC for 16 hour. The restriction enzymes digested products were electrophoresed in 2.5% agarose gel containing ethidium bromide (1%) @ 5µl /100ml by submarine gel electrophoresis apparatus using 1X TBE buffer. For 2.5% agarose, 1.5 gm agarose was taken in a conical flask and 60 ml of 1X TBE buffer was added and the gel was allowed to set in the similar way as stated in the preparation of 1.5% gel.
Table 2: Contents of PCR master mix
|Double distilled H20||17.525 μl|
|10 x PCR buffer||2.500 μl|
|MgCl2 (15 mM)||0.025 μl|
|dNTPs (10mM)||0.500 μl|
|Primers each (100 pM/μl)||0.600 μl|
|Taq DNA polymerase (5 U/μl)||0.250μl|
|Genomic DNA (50 ng/μl)||3.000 μl|
Result and Discussion
The band size were judged by comparing with molecular size marker and recorded. Genotyping of IL8 gene loci were carried out according to the band pattern of respective genotypes. The quality of the isolated DNA were depicted in Lane 1-17 are working DNA samples and Lane 18-19 are high DNA concentration (Fig.1).
Fig.1: Quality checking of Genomic DNA on 0.8% Agarose gel
The PCR-RFLP of IL8 gene by Hae III and Dra I generated monomorphic patterns in Jaffarabadi buffaloes. Exon3 revealed one band patterns for all the samples. The amplified products were visualized under UV light and documented by photography (Fig.2). Contrary to the present finding, Meade et al. (2012) reported significant different in frequencies in two divergently selected HF and Norwegian Red breeds of cattle. Hazare (2009) studied on IL-8 gene polymorphism in 179 lactating Sahiwal cattle involving whole gene including coding and non-coding regions with the product size of 500, 408, 584, 426, 418, 499, 581 & 578bp. First seven parts were monomorphic in PCR-RFLP using AluI and DraI restriction enzyme.
Fig.2: The amplified products under UV light
Similarly, Sharma et al. (2013) reported monomorphism pattern and Singh (2008) reported polymorphism in Murrah buffaloes. Since, the results were found monomorphism, the further association study was not conducted for occurrence of mastitis.
The PCR-RFLP method using Hae III and Dra I restriction enzymes in all Jaffarabadi buffaloes included in the study exhibited monomorphism in Interleukin-8 gene. The monomorphism identified in the Interleukin-8 gene in Jaffarabadi buffaloes indicated a high level of genetic uniformity in the population.