Screening And Identifying Milk Quality Traits Relative Genes Of Wenzhou Buffalo With Gene-Chip Technology

Wenzhou buffalo is a protected variety of National livestock and poultry genetic resources. compared with the ordinary milk, the buffalo milk has a higher dry matter content, with great potential value. Therefore, to explore the Wenzhou buffalo functional genes and single nucleotide polymorphism molecular markers can provide a theoretical basis and technical support for the molecular marker-assisted breeding of Wenzhou buffalo and the development and utilization of buffalo milk products.In this experiment,30Wenzhou buffaloes from Pingyang Zhiting Wenzhou buffalo Co., Ltd. and Jingmen Shayang buffalo breeding demonstration base were chosen and equally divided into3groups, early lactating period, middle lactating period and late lactating period. Peripheral blood leukocytes genome expression profiling of different lactation Wenzhou buffaloes was analyzed by Affymetrix Microarray. From the results of KEGG pathway of2times up or down differential expression genes, The2times up or down differential expression genes in the JAK-STAT pathway, TGF-beta pathway and the Wnt pathway were confirmed. According to the biological functions and molecular functions of the4differential expression genes, IL-21R gene was initially identified as one candidate gene which may be related with lactation traits.SNPs of the IL-21R gene was detected in134Wenzhou buffaloes by PCR-SSCP method. In addition, The SNPs in STAT13’UTR、 STAT4intron20and PRL exon4were also detected in the same test group.Results suggested:(1)The results of the Wenzhou buffaloes Affymetrix Microarray showed:the gene quantity and rate of the different lactating periods were early lactating period13091/54.30%, middle lactating period12187/50.50%, late lactating period11697/48.50%and dry period11967/49.60%respectively. In different lactating periods, there were772-Fold-up-change genes and742-Fold-down-change genes between early lactating period and middle lactating period, there were292-Fold-up-change genes and912-Fold-down-change genes between middle lactating period and late lactating period, there were512-Fold-up-change genes and822-Fold-down-change genes between early lactating period and late lactating period.(2)GO analysis of2-Fold-up-change genes declared, differential expression genes in biological process involved locomotion, negative regulation of biological process, positive regulation of biological process, macromolecular complex, immune system process, establishment of localization, localization, response to stimulus, developmental process, multicellular organismal process, metabolism, regulation of biological process,biological regulation, cellular process, physiological process; differential expression genes in Cellular component involved organelle part, organelle, extracellular region part, cell part, cell; Molecular function involved enzyme regulator activity, transporter activity, molecular transducer activity, binding, catalytic activity.(3) From the results of KEGG pathway of2times up or down differential expression genes, Including the JAK-STAT pathway,TGF-beta pathway and the Wnt pathway, a total of20pathways was chosen for confirming the differential expression genes,There are262-Fold-up-change or2-Fold-down-change differential expression genes including IL-21R、 Smad2、 DCN, THBS1and so on. Differential expression genes involved receptor activity, protein binding, double-stranded DNA binding transcription factor activity, the activity of the protein dimer, the calcium-binding.(4) According to the analyzation of Affymetrix Microarray, IL-21R gene which belongs to JAK-STAT pathway was chosen as the study target. SNPs of the IL-21R gene was detected in Wenzhou buffaloes by PCR-SSCP method.The experimental design21pairs of primers. The results found that7mutated sites were detected in U5, U6, U17and U19primers:A19819G,C19834T,C20357T,G20490A,C20531T,G34152A and G34523A. Using the Haploview4.2software for the IL-21R SNPs analysis and constructing two single-domain (Haplotype block). The two SNPs A19819G and C19834T are Chain mutations by the formation of single-domain1, The three SNPs C20357T、 G20490A and C20531T are Chain mutations sequence represented by the formation of single-domain2.A11of the IL-21R SNP PIC are moderate polymorphism, X2test results showed that in addition to the SNP G34523A, the other6SNPs were not in Hardy-Weinberg balance.(5) SNPs in STAT13’UTR,STAT4intron20and PRL exon4were separately amplified from gDNA obtained from Wenzhou buffaloes. The results decleared, three moderately polymorphic SNPs was detected within STAT13’ UTR,STAT4intron20and PRL exon4.