Functional Molecular Markers Of Hmgb3and Hmgb1Gene Associated With Mastitis Resistance In Dairy Cattle

Mastitis is an inflammatory disease caused by genetic and environmental factors, and many immune genes are involved in the process of mastitis. HMGBs are a non-histone nuclear protein containing HMGB1, HMGB2, HMGB3etc, and function as universal sentinels for nucleic-acid-mediated innate immune responses. Promoter, splice variants and microRNAs play important roles in the immuene response and gene expression of transcription and post-transcription regulation. To identify SNPs of mastitis resistantance as experimental basis for cow breeding, we have investigated the regulation mechanisms of HMGB3and HMGB1gene, and screened out functional SNPs associated with SCS of dairy cattle. The thesis was described as the following4sections:Section1:HMGB3plays an important role on the activation of innate immune responses. In order to explore the expression of two splice variants and relationship of the splice variant (TV1/TV2) with mastitis, the expression of the two splice variants in various bovine tissues were performed by RT-PCR, and the expression of total HMGB3RNA in healthy and infected mammary glands was investigated using Q-PCR. The results showed that the TV1was highly expressed in the MG, and the expression in H-MG was higher than that in muscle, liver and spleen of the healthy cows; While TV2was primarily expressed in liver, the expression in the mastitis mammary gland tissues was higher compared with healthy mammary gland tissues (P<0.05); HMGB3have different expression levels of in muscle, liver, kidney, spleen, lung of cow, it was highly expressed in the mammary gland, the expression of HMGB3in the mastitis-infected mammary gland (M-MG) tissues were up-regulated by8.5-fold when compared with the healthy group (P<0.05); The expression in mammary gland of the HMGB3mRNA was higher than that in muscle, liver, kidney, spleen of the healthy cows, and that is almost not expressed in lung.Section2:Promoter determines the transcription start site and transcript frequency, and controls the starting and the strength of gene expression. Howere, the property of HMGB3 promoter or the molecular mechanisms of transcriptional regulate was little known. In this study, bioinformatic analysis, gene clone, DNA sequencing, luciferase reporter gene system and PCR-RFLP methods were used to construct seven segments covering predicted promoter1,2and two fragments with different point mutation C and T in g.+2690covering predicted promoter3in the intron3as the recombinant reporter plasmids and transiently expressed in293T cells to determine the promoter activity of each fragment, respectively. The results showed the basal activity promoter1and2core sequences were mapped in the region (g.-655～g.-114) and the region (g.-116-g.+301), respectively. The promoter3with genotype TT had stronger promoter activity, and the mutation increased the expression of HMGB3gene. The associations of the SNP (g.+2690C>T) with SCS in302Chinese Holstein cows were genotyped and analyzed. It revealed that the SNP was not satisfied Hardy-Weinberg equilibrium (P<0.05). The association of different genotypes with the bovine somatic count score (SCS) was also analyzed. The findings showed that the SNP significantly affected mastitis, the SCS of cow with genotype TT in g.+2690position was significantly lower than that of genotype CC (P<0.05) or CT (P<0.05), which indicates that g.+2690C>T-TT may be a mastitis resistant genotype. These findings will also help understanding biological function of HMGB3gene in dairy cattle, and provide a new method in bovine mastitis resistance.Section3:MiRNAs are a class of small single-stranded, endogenous and non-coding small RNAs molecules (18-24nucleotides) and are post-transcriptional regulators of gene expression, either by binding to partially complementary sequences in the3’UTR to inhibit target mRNA translation into protein or accelerate its degradation. It can modulate key pathways involved in the innate and adaptive immune responses. In this study, to identify miRNAs targeting on HMGB3, we integrated the output results of multiple prediction programs:Targetscan Human, RNA22and Patrocles. We imported the various output formats from each of the target prediction programs, and subsequently integrated the results to find common overlaps:Bta-miR-17-5p, miR-20b and miR-93. The relative expression of miRNAs was determined by Q-PCR. The results showed that expression of target bta-miR-17-5p, miR-20b and miR-93of the HMGB3gene was down-regulated by1.56-,1.72-and2.94-fold in mastitis-infected cow’s mammary glands as compared with the healthy bivine tissues (P<0.05). Therefore, these three miRNAs may target on HMGB3gene, and are likely to be involved in mastitis of dairy cattle.Section4:As an important mediator of inflammation, HMGB1can participate in the inflammatory and autoimmune disease. In order to identify a SNP in the3’UTR of HMGB1gene whether affects the binding to its target miRNA or not, the expression of HMGB1mRNA in mammary glands of dairy cattle with different genotypes and its candidate bta-miR-223was investigated by Q-PCR. The results showed that the relative expression of HMGB1mRNA with genotype GG in cows was significantly higher than that with the genotype AA (P<0.05). The expression of bta-miR-223was upregulated by1.95-fold (P<0.05) in the bovine mastitis-infected mammary gland tissues compared with that in the healthy tissues. Subsequently, luciferase assay indicated that the HMGB1expression was directly targeted by bta-miR-223in293T cells. This novel SNP (g.+2776A>G) not only altered the binding of HMGB1and bta-miR-223, but also associated with SCS in cows. Taken together, the g.+2776A>G-GG could be used as a candidate functional marker for mastitis resistance breeding program.