The Study Of MBL2Gene And Dairy Cattle Mastitis Resistance

Infectious disease has major adverse effects both on the economics of livestockproduction and on animal welfare. Cow mastitis is a common disease in dairy industryworldwide，and has caused tremendous loss to the dairy production. A number oftherapeutic, prophylactic and management strategies have been proposed to minimizethis complex disease. Although these treatments have very good clinical outcome,emerge of drug-resistant strains and remaining antibiotics in milk have brought greatconcerns. Thus, an approach based on improving the host genetics in resistance toinfectious diseases through molecular marker selective breeding is becoming widelyaccepted. Mannose-binding lectin (MBL) are collagenous C-type lectins involved inthe innate immune response to various microbial pathogens. Antimicrobial functionsof MBL, including opsonization, neutralization, and complement activation. MBLselectively targets invading microorganisms for neutralization by its recognitiondomain’s binding to cell surface mannose, then activating MBL-associated serineproteases (MASPs).By screening the genetic variation of MBL2exon in825individuals of ChinaHolstein, using PCR-SSCP techniques, four new SNPs were found in exon1, g.1164G>A, g.1197C>A, g.1198G>A,1207T>C. No SNPs were found in exon2, exon3andexon4in my research. Statistical analyses revealed correlation between both g.1164G>A, g.1197C>A and somatic cell score (SCS).The effect of polymorphism g.1164G>A and g.1197C>A loci of MBL2gene oncomplement activity and serum MBL-C level was analyzed, the results indicated thatin the g.1164G>A and g.1197C>A loci, the different genotype had no significantinfluence on complement activity; in the g.1164G>A, the different genotype hadsignificant correlation on serum MBL-C level.MBL2gene was amplified using reverse transcription-polymerase chain reaction(RT-PCR). The g.1164G>A loci of MBL2was mutation by rite-directed mutagenesis.The PCR product was inserted into vector pET32a(+) to construct plasmid pET32a (+)/MBL2and pET32a(+)/MBL2-TB, then the plasmid was expressed in E.coli BL21(DE3)cell that induced by IPTG. The diversity of antibacterial activity of wild-typeand mutant was assayed by agar whole diffusion-inhibition zone method and electronmicroscope using the purified recombination protein against Sta. aureu. The resultsanalysis showed the encoding a polypeptide. SDS-PAGE and Western blotting resultsshowed that the recombinant proteins were expressed in E. coli.The purified proteinMBLC had antibacterial activity, the wild-type had stronger antibacterial activity thanmutant by Scanning electron microscope and killed pathogenic bacteria by leaking thecontent out in vitro against Sta. aureus.In this research, we cloned the2285bp DNA sequence of bovine MBL25′flanking region by LA-PCR method. The analysis of this2285bp fragment indicatedthat there were no canonical TATA boxes. No CpG island and two repetitive elementswithin promoter region were also found. Of particular interest is the fact that severalwell-documented transcription factor consensus motifs are precisely conserved in theregion immediately upstream of the MBL2exon1,including binding sites for GR、SP1,NF-1, NF-1/L, AP-1et al. Multiple of the sites for enhancer were also found. ThepEGFP-N1/2285plasmid has been constructed and transfetced into293T cells byLip2000, successful expression of EGFP gene was obtained. It implies that the2285bp of bovine MBL25′flanking region is promoter functional.To further characterize the5′flanking region, we constructed a set of luciferasereporter gene constructs containing successive5′deletions of the bovine MBL2promoter. Promoter activity analysis was performed by dual-luciferase reporter assaysystem. The core promoter of the gene was located at the+52~-85bp. Some positiveregulatory elements are located at-1189～-562and-297～-85bp. And the results alsoshowed the presence of repressor elements in this region-562～-297bp and-2237～-1549bp.In our study, three SNPs including g.905C>G, g.956C>T, g.895A>T wereexamined by sequencing and g.905C>G, g.956C>T were closely linked. Statisticalanalyses revealed correlation between both g.905C>G, g.956C>T and somatic cellscore (SCS). Based on the bioinformatics analysis, the SNPs were further surveyed.FQ-PCR was used to detect mRNA expression of MBL2gene in different tissuesand the different genotype of g.905C>G and g.956C>T, the results indicated that MBL2mRNA were expressed in all tissues examined, including heart, liver, spleen,lung, kidney, mammary gland and thyroid, the expression of mRNA in the liver wassignificantly higher than other tissues, the different genotype of g.905C>G and g.956C>T had significant correlation on mRNA expression.It could be preliminarily deduced that MBL2gene was probably a major gene ora QTL linked gene which associated with mastitis resistance traits in pig.