Studies On Genetic Variation Of C4A Gene And Its Feasibility As Molecular Marker Of Mastitis Resistance In Chinese Holstein Cattle

Complement component4(C4) is a critical component in the complement activity and protection against many bacterial pathogens because it is essential to the classical and lectin activation pathways. The serum concertration of C4is lower than that of C3, and ranks second. C4is expressed primarily in the liver, but C4mRNA has also been detected in the heart, lung, spleen, kidney, brain, thyroid and mammary gland. C4has high polymorphisms and links to the major histocompatibility complex, which shows significant association with mastitis susceptibility. Therefore, it is very important to study the genetic variation of bovine C4A gene and its association with mastitis resistance.1. Polymorphism of C4A gene and its correlation with milk production traitIn the current study, the bovine complement C4A gene was screened for polymorphisms, and the associations of these polymorphisms with the hemolytic activity of the classical pathway (CH50), serum C4levels, and milk performance traits were examined. Three novel single-nucleotide polymorphisms (g.2994A>G, g.3508A>G and g.3649G>C) were detected by DNA sequencing and PCR-RFLP in1182Chinese Holstein cows. The g.2994A>G mutation in exon10led to methionine and valine exchange at position362, whereas g.3508A>G and g.3649G>C were synonymous substitutions. The statistical analyses revealed that cows with g.2994A>G-AG and g.3649G>C-CC had significantly lower somatic cell scores (SCS, P<0.01). Homozygote cows with GAC haplotypes had the lowest SCS, whereas AAG/AAC cows had the highest. The serum concentration of C4by ELISA and the hemolytic activity and antibacterial activity of CH50were also evaluated in the current study. The serum from genetically resistant (H5H5) animals displayed a significantly higher antibacterial activity against Sta. aureus and E. coli in vitro compared with the serum from genetically susceptible H2H1(P<0.001). Moreover, no significant difference in C4level was observed in three SNPs in tested populations. The individuals with H5H7showed a higher C4level than the ones with H5H5(P<0.05).2. Alternative splicing and expression of C4A geneIn order to investigate the alternative splicing and expression of the complement component4gene (C4A) in Chinese Holstein cattle, reverse transcription and polymerase chain reaction (RT-PCR) was performed. The PCR products were cloned and sequenced. One novel splice variant was identified, namely the C4A-AS. To illustrate the molecular effects of the C4A gene on bovine mastitis, six Chinese Holstein cattle were divided into healthy (non-mastitic) and mastitic groups. Real-time quantitative PCR (qRT-PCR) revealed that the C4A-complete and C4A-AS transcripts had significant expression differences (P<0.001) in healthy cows, while there was no significant differences in the mastitic group (P=0.257). Moreover, the C4A-AS increased remarkably when mastitis developed (P=0.030). We also examined the expression of C4A-complete and C4A-AS in several tissues (liver, heart, spleen, lung, kidney, tongue and muscle). The results showed that two transcripts were expressed in all tested tissues.3. Transcriptional regulations of C4A geneThe5’flanking region of the bovine gene for the fourth component of complement was sequenced and analyzed in hepatic cell lines (HepG2) and kidney cell (293T). A series of KpnI/Xhol fragments of the C4A promoter were cloned into the luciferase reporter pGL3-Basic vector using five primers, which progressively closer to the translation starting site of C4A gene. Transient transfection analysis was used to identify a169bp minimal promoter fragment from authentic initiation site which was able to direct basal transcription of bovine C4. Moreover, three cis-acting elements, SP1(-169to-158), E-box (-122to-117) and AP-1(-80to-71) were detected and proved to be essential for C4basal expression. Single nucleotide polymorphism (g.+147G>A) of intron1had effect on serum C4concentration, and may modulate the transcriptional regulation of the fourth component of complement. We also mapped the position of an LPS responsive factor in the293T cell line. LPS was able to increase transcription activity to10.0±0.06, while the activity significantly decreased with mutation in the AP-1motif. We concluded that AP-1was the critical factor responsible for LPS induction of bovine C4. Super-shift EMSA was conducted and the results showed that these transcriptional elements were existed in the HepG2cell line.In conclusions, C4A gene can serve as a potential marker of the genetically resistance of the mammary gland. The resistance breeding is merely a part of work of treating bovine mastitis. Meanwhile, management reinforce and nutrient balance are also needed in dairy cattle breeding.