A2M Molecular Mechanism Of Transcriptional Regulation And Functional Molecular Marker Screening In Dairy Cattle

Mastitis is an inflammation of bovine mammary glands with high infected rate and largedamage, which has become the main disease in dairy farming. The relative expression of A2Mprotein in mastitis-infected mammary gland was upregulated3.5fold compared with that inhealthy gland using the isobaric tags for relative and absolute quantitation （iTRAQ） method. TheA2M acts as a non-specific protease inhibitor reducing the damage and enhancing diseaseresistance of host by inactiving proteases and toxins secreted by pathogens. By means ofmolecular biology, it has important significance for bovine mastitis resistance molecularbreeding that functional molecular marker screening and transcriptional regulation molecularmechanism research of this gene was studied.1. A2M gene quantitation, identification of splicing variants and functional SNPsFor preliminary research of A2M gene in the role of dairy cow mastitis, RT-qPCR was usedto determine the expression level of A2M mRNA in mammary tissues, finding that theexpression was significantly higher in mastitis-infected mammary tissues than that in healthytissues （P<0.05）. Pre-mRNA alternative splicing can alter the sequence of gene coding region,produce a variety of proteins, and change gene functions. RT-PCR was used finding24newsplicing variants and two functional single nucleotide polymorphisms （SNPs） located in theputative exonic splice enhancer （ESE） by sequencing, c.3535A>T and c.4520T>C. Themutation of c.3535A>T increases two binding sites for the splicing factor SC35（SRSF2） andSRp40（SRSF5）, which may be involved in the splicing process of A2M-AS4; The mutation of c.4520T>C increases the splicing factor SRp40being involved in the splicing process ofA2M-AS24.2. Relevant research between A2M3’UTR and bta-miRNA-2898Mature miRNAs are known to regulate gene expression in the post-transcriptional processby binding to the3’ untranslated regions （3’UTR） of target gene mRNAs. The result showed thatbta-miR-2898can bind to the A2M gene3’UTR in the dairy cow by RNAHYBRID and RNA22 softwares, and that a SNP （c.4659₄661delC） is located within the region of miRNA-bindingsites, showing wild type （//） having lower Somatic Cell Score （SCS） through genotyping. Wildtype （CC） and mutant type （//） of3’UTR are respectively constructed into the PMIR-ReportTMluciferase report vector, transfecting293T cells with miR-2898expression vector. Through dualluciferase report system analysis, the result showed that miR-2898can bind to the A2M3’UTRreducing the expression of reporter gene, and that the c.4659₄661delC mutation has effects onthe binding affinity. These data indicate that the miR-2898binding to the A2M3’UTR couldinfluence A2M expression and may be involved in the immune function on mastitis in dairy cow.Mutant type （//） of3’UTR can be used as functional molecular markers for bovine mastitisresistance breeding.3. IdentQification of core promoter and functional SNPs in A2M genePromoter is an important part of the gene, which can control the start time of genetranscription and the degree of expression. SNPs within the5’ flankling region can influence theactivity of the promoter, and change the expression of gene. Currently, there is no report aboutpromoter region of the A2M gene in dairy cow and its corresponding characteristics. Firstlyusing bioinformatics methods to predict gene promoter region, and then constructing differentfragment length into pGL3-Basic luciferase report vector, transient transfection293T cells, lastlyg.-2641-g.-2479in5’ flanking region was identified as core promoter of the A2M gene. AP-1,E2F and TATA box of transcription factor binding sites were found in this region with thebioinformatics analysis, which may play a positive or negative regulatory role in the process ofA2M gene transcription.5’ flanking region in the A2M gene existing four SNPs （g.-724A>G, g.-665G>A, g.-535C>G, g.-520_-519insA） by sequencing, promoter fragment containing mutanttype （AAGGCC//） and wild type （GGAAGGAA） were separately constructed into the luciferasereport vector, finding the activity of wild type is significantly higher than that of wild type （P<0.05） after transfecting cells. g.-724A>G increased NKx-2.5transcription factor binding sites,and g.-520_-519insA increased C/EBPb binding sites by software analysis. Five SNPs found inthe control region were carried out haplotype combination in this study, and correlation analysisbetween different haplotype and SCS showed that haplotype combination H1H2, H1H3andH2H4（H1: AGC//，H2: AGC/C，H3: GAGA/，H4: GAGAC） has lower SCS.