Establishment Of Extraction DNA From Milk And Study On Two Candidate Genes Polymorphism In Chinese Holstein

Dairy cattle are economical animals.In order to avoid stress and harm,such as capture,handling and venipuncture during making samples collection,we established somatic cells in milk were used as a source of DNA and as a substrate for the polymerase chain reaction.Milk production traits are typical quantitative characteristics controlled by a number of genes.Secretion of fat globule in milk is primarily based on getting proper shape of fat droplets with a group of membraneous proteins of which butyrophilin(BTN) plays the pivotal role during the process of lactogenesis.As BTN gene is primarily responsible for size of fat globules by conferring major contribution towards the fat globule membrane, this gene product may be the crucial factor for determining the quantity as well as quality of milk fat production.In this study it was to estimate the relations between the the SNPs of bovine butyrophilin(BBTN1A1) gene in exon 1 and exon 3 and milk performance traits (305 days yields of milk,fat percent and protein percent as well as SCS and ME) in Chinese Holstein cows.Heat stress is detrimental to dairy production and affects numerous variables including feed intake and milk production.Heat shock protein(HSP) is a group of specific proteins that produce under stressing,which can let organism quickly adapt to environmental changes.HSP is the most important molecular chaperone involved in heat stress response of animals.In this study,it was to estimate the relations between the the SNP of HSPA1B gene and milk performance traits(305 days milk yield,fat percent and protein percent as well as SCS and ME) in Chinese Holstein cows.The results were as follows:1.We established the method of genomic DNA extraction.The patent of this method was applied.The patent ID was 200810115822.9.2.The linear regression equation Y=4.54X-1.44,the correlation coefficient r=0.98 were established between DNA content and somatic cell counts.Successful 1μg DNA extraction required a minimum total of 17×10~6 cells in the milk sample.3.The polymorphisms of BBTN1A1 gene in exon 1 in 502 Chinese Holstein dairy cows were studied.The DNA from milk samples was as for template of PCR.We used the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) technique to screen for DNA polymorphism in all cattle.We amplified an 578bp fragment of BBTNIA1 gene consisting of part of exon 1.The amplified fragment digested with TaqI restriction endonuclease and subjected to electrophoretic separation in ethidium bromid-strained 2%agarose gel.Two alleles were observed,A and B.This gave Allele frequencies of 0.7131 and 0.2869 for A and B alleles.Three genotypes were observed, Frequencies were 0.4502,0.5259,0.0239 for AA,AB and BB,respectively.DNA sequencing was showed that The T -＞C mutation at 334bp and T -＞A mutation at 336bp were found.Chi-square test showed this loci wasn’t at Hardy-Weinberg disequilibrium in Chinese Holstein.Associations were studied between the SNP of BBTN1A1 gene in exon 1 and milk production traits.It had been showed significant difference with 305 days milk yield(P＜0.05),fat percent and protein percent as well as SCS and ME(P＜0.01)between AA and AB genotype.4.We used the PCR-RFLP technique to screen for DNA polymorphism in 287 Chinese dairy cattle.In all cattle,we amplified a 682 bp fragment of BBTN1A1 gene consisting of part of exon 3.The amplified fragment digested with MboI restriction endonuclease and subjected to electrophoretic separation in ethidium bromid-strained 2% agarose gel.Two alleles were observed,A and B.This gave Allele frequencies of 0.1132 and 0.8868 for A and B alleles.Three genotypes were observed,Frequencies were 0.0105, 0.2055,0.7840 for AA,AB and BB,respectively.DNA sequencing was showed that The G -＞A mutation at 381bp was found.Chi-square test showed this loci was at Hardy-Weinberg disequilibrium in Chinese Holstein.Associations were studied between the SNP of BBTN1A1 gene in exon 3 and milk production traits.It had been showed significant difference with 305 days milk yield(P＜0.05) and ME(P＜0.01)between AB and BB genotype.5.We used the PCR-RFLP technique to screen for DNA polymorphism in 185 Chinese dairy cattle.In all cattle,we amplified a 995 bp fragment of HSPA1B gene.The amplified fragment digested with SpⅡrestriction endonuclease and subjected to electrophoretic separation in ethidium bromid-strained 2%agarose gel.Two alleles were observed,A and B.This gave Allele frequencies of 0.3946 and 0.6504 for A and B alleles.Three genotypes were observed,Frequencies were0.0865,0.6162,0.2973 for AA, AB and BB,respectively.DNA sequencing was showed that The C -＞T mutation at 881bp was found.Chi-square test showed this loci was at Hardy-Weinberg disequilibrium in Chinese Holstein.Associations were studied between the SNP of HSPA1B gene and milk production traits.It had been showed significant difference with fat percent(P＜0.05) between AB and BB genotype.