藏猪甲基转移酶3a基因的SNP筛选及其在精子中的表达分析

SNP Screening of DNA Methyltransferase 3a Gene in Tibetan Pig and Its Expression in Spermatozoa

为筛选藏猪DNA甲基转移酶3a(Dnmt3a)基因的单核苷酸多态性(SNP),分析Dnmt3a基因在藏猪精子中的表达水平,采用PCR-直接测序法对藏猪、大白猪、滇南小耳猪Dnmt3a基因进行SNP分析;分别应用实时荧光定量PCR技术、Westen blot方法对藏猪、大白猪精子中Dnmt3a基因的mRNA与蛋白质表达水平进行检测。结果显示,在Dnmt3a基因5′侧翼和第11外显子区发现2个SNP位点,位于藏猪Dnmt3a基因的5′侧翼区起始密码子上游2012 bp处的SNP(C→T)位点,其杂合度、多态信息含量均较高,表明群体内变异程度大、能提供的遗传信息较多,但该位点不是藏猪特有的SNP;位于藏猪Dnmt3a基因的第11外显子区第1360 bp处的SNP(C→T)位点,其杂合度、多态信息含量均较低,表明群体内变异程度小、能提供的遗传信息较少。藏猪精子中,Dnmt3a基因mRNA与蛋白质水平上的相对表达量均显著(P<0.05)低于大白猪。以上结果表明,藏猪Dnmt3a基因5′侧翼区起始密码子上游2012 bp的突变位点可以作为藏猪分子育种中的候选标志;相对于大白猪,Dnmt3a基因在藏猪精子中mRNA及蛋白质水平上的低表达可能是藏猪精子高原适应性的DNA甲基化机制之一。

In order to screen the single nucleotide polymorphism(SNP)of DNA methyltransferase 3a(Dnmt3a)gene in Tibetan pig and analyze the expression level of this gene in Tibetan pig sperm,SNP of the gene was analyzed in Tibetan pig,Yarkshire pig and Diannan small-ear pig by PCR-direct sequencing,and it′s mRNA and protein expression levels were respectively measured in spermatozoa of Tibetan pig and Yarkshire pig by using real-time fluorescence quantitative PCR and Westen blot.The results showed that there were 2 SNPs in Dnmt3a gene 5′-flanking sequences and exon 11 region.One SNP(C→T)site was found at 2012 bp upstream of the initial codon of Dnmt3a gene 5′-flanking sequences in Tibetan pigs,which had high heterozygosity and polymorphism information content,indicating there was large degree of variation within the population and more genetic information provided.However,this mutation site was not a peculiar SNP to Tibetan pig.The other SNP(C→T)site was found at 1360 bp of exon regio.This SNP site had lower heterozygosity and polymorphic information content,indicating that the degree of variation within the population was smaller,and the genetic information provided was less.Both the mRNA and protein expression levels of Dnmt3a gene in spermatozoa of Tibetan pigs were significantly lower(P<0.05)than that of Yarkshire pig.The above results showed that the mutation site of 2012 bp upstream of the initial codon in the 5′-flanking region of Dnmt3a gene in Tibetan pig could be used as a candidate marker in the molecular breeding.It might be one of the DNA methylation mechanism of Tibetan pig sperm plateau adaptation that there were lower mRNA and protein expression levels of Dnmt3a gene in spermatozoa of Tibetan pig compared to Yarkshire pig.