Variation Of MASP-2 And Its Associations With Mastitis In Chinese Holstein Cattle

Mannan binding lectin-associated serine protease 2 (MASP-2) is a key factor of the lectinpathway of complement activation and play an important role in the defense. MASP-2 is expressedprimarily in the liver, it is complexed with mannan binding lectin (MBL) or ficolin, havingbiological functions. Therefore, it is very essential to study the genetic variation of bovineMASP-2 gene and its association with mastitis resistance.1. Polymorphism of MASP-2 gene and its correlation with milk production traitIn the present study, we discovered five single nucleotide polymorphisms of the MASP-2gene by screening the genetic variation in 759 individuals using DNA sequencing techniques,created restriction site polymerase chain reaction (CRS–PCR), and PCR restriction fragmentlength polymorphism (PCR–RFLP) and analyzed their associations with milk traits. The fivesingle nucleotide polymorphisms were g.-282 G>A,g.478 G>A,g.506 G>A,g.511 T>C andg.13808 G>A. One SNP (g.-282 G>A) was located in 5’- flanking regions and three SNPs (g.478G>A,g.506 G>A,g.511 T>C) in exon III, and g.13808 G>A was located in exon XI. g.478 G>Aand g.511 T>C were synonymous mutation,g.506 G>A and g.13808 G>A were non-synonymousmutation . Three of the SNPs (g.-282 G>A, g.478 G>A, g.511 T>C) were firstly found to belinked completely and regarded as a SNP g.511 T>C. The statistical analyses revealed that thecombined genotypes of H1H8 with the lowest SCS and the highest protein content, H1H4 withthe highest fat content, and H1H2 with the highest 305 d milk yield were favorable combinationsfor mastitis resistance.2. Alternative splicing and mRNA expression analysis of MASP-2 geneBased the result of diagnostic tests for identification of mastitis and the data of SCS, seletedsix bovine tissues, divided into two groups: normal ( n = 3), mastitis ( n = 3).We characterizedfour novel bovine MASP-2 splice variants by reverse transcription and polymerase chainreaction (RT-PCR), designated as MASP-2 A125，MASP-2 A145，MASP-2 A25，MASP-2 A245.All four novel MASP-2 isoforms are derived from the complete transcripts (MASP-2 complete)via alternative splicing (AS). The patterns of the four splice variants are intron retention andexon skipping. In the present study, the detected frequency of four splice variants was 22.9% outof the 35 sequenced clones. All transcripts of MASP-2 were mainly expressed in hepatocytes.We found MASP-2 complete was the main transcripts in mastitis bovine and the levels ofMASP-2 complete, MASP-2 A25 in the normal hepatocyte tissues were higher than other transcripts.3. Cloning and Identification of Core Area of MASP-2 Gene PromoterIn this research, the 5’ flanking region of the bovine gene including the 5’ UTR(untranslation region) and partial coding sequence was cloned. The sequence was analyzed inhepatic cell lines (HepG2) and kidney cell (293T) respectively. There is promoter activity in bothHepG2 and 293T cell, but the promoter activity in HepG2 cell is stronger than in 293T cell. Thestudies showed that the sequence from +245—+577 bp had the basal promoter activity in HepG2cell. There were positive (-1136—-666 bp and-244—+245 bp) and negative (-1915—1136 bpand-666—-244 bp) regulatory domains, respectively.