三河牛β-乳球蛋白基因型及其启动子序列特点

Genotype and promoter sequence characteristics ofβ-lactoglobulin in Sanhe cow

[目的]分析三河牛β-乳球蛋白(β-Lg)的基因型,并探索其启动子序列与乳中含量的关系。[方法]采用聚丙烯酰胺凝胶电泳(PAGE)检测乳中β-Lg遗传变异体及其相对表达量,并通过PCR扩增,比对不同遗传变异体启动子的序列差异。[结果]三河牛(n=62)乳清中β-Lg有AA、AB和BB共3种基因型,频率依次为0.290、0.403和0.307,A和B两种等位基因频率接近;对37个纯合型β-Lg启动子的部分序列进行PCR扩增(636 bp)和测序,并与普通牛β-Lg基因序列(GenBank登录号:U31361.1)比对,共检测到6个突变位点,其中-430、-362、-216和-204位点基本上是等位基因特异的,且有3个突变点在3种转录因子结合基序中;建立了三河牛乳中β-Lg含量的高效液相色谱测定方法,发现β-Lg含量在AA型中极显著高于AB型(P<0.01),但AA、AB型与BB型之间均无显著差异。[结论]三河牛β-Lg A和B两种等位基因频率接近,其启动子-430、-362、-216和-204位点具有等位基因特异性,并可能影响等位基因表达。

[Objective]The genotype ofβ-lactoglobulin(β-Lg)in Sanhe cow was analyzed,and the relationship between its promoter sequence and milk content was explored.[Method]Polyacrylamide gel electrophoresis(PAGE)was used to detect the genetic variants ofβ-Lg in milk and their relative expression levels,and the sequence differences of different genetic variants were compared by PCR amplification and sequencing.[Result]There were three genotypes of AA,AB and BB inβ-Lg in whey of Sanhe cows(n=62),and the frequencies were 0.290,0.403 and 0.307,respectively.The frequencies of A and B alleles were similar.The partial sequences of 37 homozygousβ-Lg promoters were amplified by PCR(636 bp)and sequenced,and compared with the bovineβ-Lg gene sequence(GenBank accession number:U31361.1).A total of 6 mutation sites were detected,of which the-430,-362,-216 and-204 sites were basically allele-specific.A HPLC method for the determination ofβ-Lg in Sanhe cow milk was established.It was found that the content ofβ-Lg in AA type was significantly higher than that in AB type(P<0.01),but there was no significant difference among AA,AB and BB types.[Conclusion]The allele frequencies ofβ-LgA and B in Sanhe cow were similar,and the promoter-430,-362,-216 and-204 loci have allele specificity and may affect allele expression.