DNA甲基转移酶1基因的克隆测序及其在牦牛和犏牛睾丸组织中的表达

Cloning and sequencing of DNA methyltransferase 1 gene and its expression in testis of yaks and cattle-yaks

为了研究牦牛DNA甲基化转移酶1(DNA Methyltransferase 1,DNMT1)基因,并比较其在牦牛、犏牛睾丸组织中的mRNA水平,试验以成年麦洼牦牛睾丸组织cDNA为模板,利用RT-PCR扩增DNMT1基因,通过连接、转化等过程克隆出牦牛DNMT1基因CDS序列;并对其编码的蛋白质进行生物信息学分析、蛋白质相互作用网络预测分析;采用实时荧光定量PCR方法检测在牦牛(n=10)、犏牛(n=10)睾丸组织中DNMT1 mRNA的相对表达量。结果表明:牦牛DNMT1基因CDS全长为4836 bp,编码1610个氨基酸;牦牛DNMT1基因CDS序列与多胃反刍动物的同源性较高(>96%)。牦牛DNMT1蛋白分子质量为182.7 ku,无跨膜结构,无信号肽,为带正电荷的亲水性非分泌蛋白;DNMT1蛋白的二级结构主要由无规则卷曲和α-螺旋构成;STRING相互作用网络显示其与DNMT3A、DNMT3B、组蛋白H3蛋白共表达;在犏牛睾丸组织中DNMT1 mRNA的表达极显著低于牦牛(P<0.01)。说明DNMT1基因可能会影响犏牛雄性不育。

In order to study the DNA Methyltransferase 1(DNMT1)gene of yak and compare its mRNA level in yak and cattle-yak testis tissue,the experiment took adult Maiwa yak testis tissue cDNA as template,using RT-PCR to amplify the DNMT1 gene,and to clone the CDS sequence of the yak DNMT1 gene through ligation,transformation and other processes.Real-time fluorescence quantitative PCR was used to detect the relative expression of DNMT1 in the testis of yak(n=10)and cattle-yak(n=10).The results showed that the CDS region of the yak DNMT1 gene was 4836 bp in length,encoding 1610 amino acids;the sequence of the CDS region of the yak DNMT1 gene had a high homology(>96%)with that of polygastric ruminants.The yak DNMT1 protein had a molecular mass of 182.7 ku,no transmembrane structure,no signal peptide,and was a positively charged hydrophilic non-secreted protein;the secondary structure of DNMT1 protein was mainly composed of random coils andα-helices;STRING interaction network showed that it was co-expressed with DNMT3 A,DNMT3 B and histone H3 proteins.The expression of DNMT1 mRNA in the testis tissue of cattle-yak was significantly lower than that of yak(P<0.01).The results suggested that DNMT1 gene may affect the male sterility of cattle-yaks.