The Mechanism Of Complement C6-C9Gene In Mastitis Of The Dairy Cattle

The productive traits of the cattle is a very important economic indicator in dairyproduction. The cost and the level efficiency in the production process depends on thelevel of the reproductive performance directly. Heritability of the cattle as animportant criterion measure reproductive traits that is affected by the main factors:genetic, environmental, nutritional, disease and so on. Among them the disease factoris particularly fecundity. Mastitis is common disease in catties multiple risk factorswhich mainly caused by the grounds trauma, improper operation of milking, thesurrounding environment, hormone imbalance, breast defects or a host of otherdiseases induced by pathogen invasion infection. The characteristics of mastitiscomplexity, diversity as well as refractory. The mastitis incidence rate in China ishigher than in Europe and America, while the annual economic losses caused by over50million and still increased in China. The diagnosis of the recessive mastitis mainlyby detection of somatic cell count (SCC) in milk samples. The numerical value ofSCC is an important feature of the degree about the health and milk quality. Theconventional control means measured for recessive mastitis are used of antibiotics butcould easily lead to drug residues. At the same time, the low fecundity and longgeneration interval of the cattle cause too long and ineiffcient of the target cycle.Currently a hot research derection is regjalation with mastitis in the level of geneassociated, that is one of the effective techniques. The complement system are present in the plasma and the complexity of theinteractions on the cell surface proteins, as a signiifcant humoral immune effector ofthe innate immune mechanisms regulatory system. The complement system canidentiifed, bind to and kill invading microorganisms. The three activation pathway ofthe complement system: the classical pathway, lectin and alternative pathways crosswith each other to play the effect of three ways eventually，and then lead to theformation of membrane attack complex (MAC). MAC gathered in the surface of thetarget cell-mediated cytolytic effect, so that the the transmembrane perforation to killthe target cells. Complement C6-9are the most important component of the terminalpathways which play an important role in the defense mechanisms of autoimmune andimmune surveillance. In summary we predict Complemnet C6-9gene might beinterested in and effected the mastitis. In this experiment, we studied the exon sites ofC6gene in coding region polymorphisms and mastitis，relevance that whether the C6gene could be used as a candidate gene for resistance to mastitis.AS (Alternative splicing AS) is the process of different type isoform whichgenerated from the same mRNA precursor (Pre-mRNA). Alternative splicing encodeplurality different transcripts, and then translated into different kind of proteinsequence and structure which thereby can lead to a diversity of protein function. Onthe level of the entire human genome analysis, we have found the different genecontain rich forms of alternative splicing. So that we conclude that the complementC6-9gene of the cattle has different splicings，and then can make more detailedanalysis on the expression of RNA in normal and suffering from mastitisorganizations. The ifnal product of gene expression is RNA and protein, and both of those secondary substances maintained methodical activity of the life with them.Therefore, the mechanism of regjalation of gene expression that has been a hotresearch and cutting-edge plays a key role in the regjalation of the promoter in genetranscription links. In the present study, we predict the core promoter region range ofC6-9gene，iflter the promoter region of the presence of SNPs，and analysis theassociation C9gene SNPs with the promoter core area.1. C6gene polymorphism and correlation analysis of dairy production traitsIn this study, we use DNA sequencing and PCR-RFLP method to detect thesingle nucleotide polymorphisms sites (SNPs) of C6gene in469Holstein catties. Wehave found more SNPs genotyping, researched the different alleles and genotypeswihich associated genetic correlation analysis of different haplotypes and haplotypecombination with cattle production traits. Also we have screened molecular markerswhich related with dairy production milk traits and mastitis to provid a reference forthe development of high yielding milk, high quality milk protein and milk fat catties.We found four SNPs: g.+2235G〉A mutation in exon2，g+32881C〉A andg.+32925C〉T mutations in exon8, g+69060T〉C mutation in17exons aftersequence analysis. Groups tested consisted of four SNPs into16haplotypes, in whichthere are10haplotype combination according to the statistical analysis. By SASsoftware statistical analysis, we found that as follows: in groups with g+32881C> Amutation, somatic cell count of CA genotype was signiifcantly higher than the CCgenotype (P<0.5)，and milk yield was signiifcantly higher than the AG individuals(P<0.05); in groups with g.+69008T> C mutation, the individual milk yield of the CCgenotype was signiifcantly higher than the TT genotype (P <0.01)，meanwhile the milk protein was signiifcantly higher than the TT genotype (P <0.05). The other twopoint mutations in the popjalation studied traits was no signiifcant difference (P>0.05).The H1H9(GCCT/ACCT) was found to be the best mastitis resistance and haplotypecombination of high milk production, while somatic cell score of the H2H2(GCCC/GCCC) was signiifcantly higher than other haplotype combination as a susceptibilitygroups.2. the identification of alternative splicing in C6-9geneAccording to the level of somatic cell score and milk pathogen identiifcationresults, the eight catties sample was divided into health groups and mastitis groups.The C6，C7，and C9gene transcripts diiferent from the original gene sequence werefound in the liver, breast, and spleen tissues by RT-PCR，QRT-PCR and cloningtechnology. They are named C6-AS1，C7-AS1，C7-AS2，C9-AS1，while the originaltranscription of C6，C7and C9were named C6-complete, C7-complete, andC9-complete respectively. The splicing mode of C6-AS1is the5,6exon completeabsence; the C7-AS1is completely absenced of2-8exon with intron completelyretained, the6，7intron and exon7of the C7-AS2is completely missing; and theC9-AS1splicing of exon6partial defects. Three different gene transcripts wereexpressed in the liver and breast.Prediction of protein functional domains show the C6，C7，C9occurrence ofalternative splicing and protein domain changed greatly. So that we predict theactivity may be changed following these alternative splicings. The test by real-timequantitative research on mRNA expression of C6，C7，C9gene transcripts in healthygroup and the group of mastitis has shown: C6-complete transcription of theVIII organization of the health organizations and mastitis organizations differentiallyexpressed signiifcantly(P<0.05)，while the C6-AS1transcriptions of the twoorganizations express signiifcantly ddifferently(P<0.01), the two transcriptions of theexpression differences in healthy tissue signiifcant (P<0.05). The C7-AS1transcription of the expression in the two tissues were signiifcantly different (P<0.05)，the C7-complete and the C7-AS1transcription of the expression of the difference wassigniifcant (P <0.05) in normal tissues. As to C9gene，the two transcripts expressedno signiifcant different in the two tissues.3. identification and analysis of the core promoter region in C6-9geneBioinformatics of the5’ regulatory region of C6-9gene was predicted by onlinesoftware. Also we choice reference to design appropriate primers and thensuccessfully constructed a series of vectors transfected into HepG2cells. TheDual-Luciferase Reporter Assay System was used to analysis the promoter activity ofthe5’ lfanking region of C9gene in the cattle. A SNP loci (g.-2059C〉T) wasidentiifed by sequencing, and the promoter activity impacted by the SNPs wasanalysed. The results shows the promoter of the C6，C7and C8were absence amongthe sequence-2500. It is detected the core area promoter of C9gene was g.-526?g.+4on consistent with the predicted results. The study with g.-2059C>T of thecorrelation between genotype and SCS shows it is not significant, also we found theconcentration of C9was not a signiifcant change.In summary, the complement C6-9were chosen as mastitis resistance candidategenes by the methods of RT-PCR，qRT-PCR，PCR-RFLP, sequencing, Dual Luciferasedetection and gene screening target genetic polymorphism which combined with the cattle’s production performance measurement data in this study. By analyzing of therelationship between genetic polymorphisms of candidate genes and mastitis, weinitially contained validation of candidate genetic polymorphism and found a possiblemastitis resistance genotypes. We obtained validation of different transcripts encodingamino acids, suggested the role of alternative splicing of protein activity. The differentlength of the promoter region of the carrier transfected cell lines was veriifed the coreregion of the promoter, and its function were described to lay a theoretical foundationfor marker-assisted selection with mastitis resistance.