PCR-SSCP Analysis On The Partial Sequence Of BLF Gene And The Exploration Of Its Feasibility As Molecular Marker Of Mastitis Resistance

The aim of this paper is to characterize the relevance between mastitis resistance and the variants of bovine lactoferrin gene by the PCR-SSCP analysis of bLF partial sequence, to search the putative transcription factor binding sites in the 5' flanking region using the bioinformatic methods, and to predict the feasibility of these variants as molecular marker of mastitis resistance combined with the CMR data and other production records.1 The Isolation of DNA from Cow MilkIntegrating the method of isolation of leukocytes from milk and the method of isolation DNA from the cells, the method of isolation of DNA from cow milk was built. The results indicated that the average concentration of isolated DNA stock solution was 0.5 μg/ml, and the relative value of OD₂₆₀ /OD₂₈₀ was 0.9 which demonstrated that the remnant of protein was too high. But through the analysis of PCR, the DNA stock solution reached the requirement of PCR reaction, which indicated that this method could be an alternative method to isolate DNA.2 The PCR-SSCP analysis on the partial sequence of bLFFour fragments within 5' flanking region and two fragments of exons 4,15 were amplified using the sequences registered in the NCBI to design the six pairs of primer. And using the PCR-SSCP, the nucleotide diversity was identified and these variants were sequenced. The putative transcription factor binding sites within 5' flanking region were searched by the bioinformatic methods. The results indicated that for four fragments within 5'-region: P1, P2, P3, P 4 from upstream to downstream, we found that two, P1 and P4, had four base variations, -945 site T→C, -928 site G→A, -28 site A→C, +33 site C→G; exons 4 and 15 have two mutations, 118 site A→G and166 site T→C, in which the mutation of 118 site A→G resulted in the amino acids mutation Ile→Val; Analyzed by DNAStar software, the amino acids mutations had no effects on the bLF protein in hydrophobicity and isoelectric point; many putative transcription factor binding sites within 5' flanking region were found...