Mechanism Of Experss Regulation And Function Of Clusterin Gene In Dairy Cow Mastitis

Bovine mastitis is a breast lesion caused by physical, chemical, or microbial stimulation. In cows, it can cause huge losses in milk production and is therefore of great importance in dairy farming. Although the symptoms and methods for prevention of mastitis in dairy cows have been studied for nearly a century with some effect, its pathogenic mechanisms are still largely unknown and thus the problem cannot be completely controlled. Here, we analyzed the role of clusterin as a potential mastitis resistance gene. We investigated the molecular mechanisms of clusterin gene regulation and its role in mastitis and milk production traits.1Detection of single nucleotide polymorphisms (SNPs) in the bovine clusterin gene and their association with mastitis and milk production traitsIn this study, we analyzed the association of the clusterin (CLU) gene with bovine mastitis and milk traits. By sequencing, we detected SNPs in the5’flanking region, coding region, and3’untranslated region (UTR) of the CLU gene. We then analyzed the identified polymorphisms and their haplotypes, as well as birth order, for association with mastitis and the milk production traits of more than1000Chinese Holstein cows. The milk production traits analyzed were:milk yield, milk fat, milk protein percentage, somatic cell count, lipoprotein ratio, total milk volume, total milk fat, total milk protein, and milk performance. We found that, although there is little genetic variation among Chinese Holstein cows, a+17301T deletion in the CLU gene3’UTR was associated with mastitis and could therefore be used as a marker. The previously reported haplotype HH4was not associated with any trait. The birth order effect was important:somatic cell count increased with more previous births and milk performance stopped improving after the fourth birth. 2In silico and functional analysis of the clusterin promoterNext, we analyzed the5’flanking region of the bovine CLU gene by bioinformatics. We then cloned different fragments containing promoter (TATA box) fragments, transcription factor binding sites, and/or CpG islands into the pGL3-Basic luciferase expression vector. We then analyzed promoter activity after transfection into293T cells. We found that the F1promoter had very weak activity, and that the C994T polymorphism within it had no notable effect. However, the F2promoter had strong activity. We then generated enhanced green fluorescent protein (EGFP)-based expression vectors with different F2-related promoters (pEGFP-N1-CMV-CLU, pEGFP-N1-F2-CLU, and pEGFP-N1-CMV-F2-CLU) and transfected them into293T cells and mammary epithelial cells. We found that both the pEGFP-N1-F2-CLU and pEGFP-N1-CMV-F2-CLU constructs could stimulate high levels of CLU gene expression and activity in293T cells. Expression of the composite promoter vector, pEGFP-N1-CMV-F2-CLU, activated CLU gene expression the strongest, and we observed expressed CLU protein in the cytoplasm.3Alternative splicing and expression of the clusterin gene in healthy and mastitis-infected mammary tissue in Chinese Holstein cowsWe cloned and sequenced the CLU mRNAs identified in mastitis-affected mammary tissue to examine alternative splicing. We identified two alternative splice variants, X-CLU and Y-CLU. Moreover, a SNP in the coding region led to the loss of many shear sites and caused dramatic changes in the CLU mRNA secondary structure. Both the X-CLU and Y-CLU alternative splice variants were expressed in vivo in normal and mastitis-affected breast tissue, but the expression of X-CLU was increased significantly in mastitis. At the protein level, an80-kDa CLU band was present in normal and mastitis-affected breast tissue (n=6each) and a60-kDa band was observed in one sample of each. When we generated constructs specifically expressing either splice form (pEGFP-N1-CMV-F2-X-CLU and pEGFP-N1-CMV-F2-Y-CLU) and expressed them in mammary epithelial cells, we could detect expression of the X-CLU mRNA and the60-kDa protein, but not the Y-CLU mRNA or the80-kDa band. The Y-CLU-derived,80-kDa protein may be expressed only in certain environmental conditions, and may form protein complexes with the X-CLU-derived60-kDa protein. 4A clusterin SNP affecting microRNA binding is associated with mastitis in dairy cattleOf the three variants we identified the3’UTR of the CLU gene, bioinformatics analysis of the mastitis-associated+17301T deletion suggested that this site may play an important role in microRNA (miRNA) binding. We found that the normal allele bound to bta-miR-2373, but that this binding was abolished in the presence of the+17301T deletion. Moreover, we demonstrated by gene-chip analysis that bta-miR-2373was downregulated four-fold in mastitis-affected breast tissue from Chinese Holstein cattle compared with in non-affected tissue. Study found that MicroRNA precursor expression cloning vector CCS-bta2373-MR04in293T cells overexpress, CLU gene3’UTR of target expression cloning vector pMIR-REPORT-miRNA-PT is almost can be fully combined with CCS-bta2373-MR04completely inhibit the bta-miR-2373expression, pMIR-REPORT-miRNA-PT-SNP can also be part of the combination CCS-bta2373-MR04and reduce the bta-miR-2373expression, resulting in mastitis pathogenesis in the up-regulation of CLU gene expression, participation in mastitis occurrence and development. We propose that the expression level of bta-miR-2373can be used as a mastitis resistance breeding selection marker in dairy cows.