牦牛DDAH1和DDAH2基因的克隆测序及其在牦牛和犏牛睾丸中的表达差异

Cloning and sequencing of DDAH1 and DDAH2 genes in yaks and their differential expressions in the testes of yaks and cattle-yaks

为了测定牦牛DDAH1和DDAH2基因序列并比较其在牦牛和雄性不育犏牛睾丸中的表达,以探究该基因与犏牛雄性不育的联系,试验从牦牛睾丸中提取总RNA,采用RT-PCR技术克隆并测序获得牦牛DDAH1和DDAH2基因的c DNA序列;利用实时荧光定量RT-PCR技术检测这两个基因在牦牛与犏牛睾丸中的表达。结果表明:克隆获得的牦牛DDAH1和DDAH2基因序列分别长959 bp及1 091 bp,均包含858 bp的CDS区。DDAH1基因序列与普通牛相比有4个碱基差异,序列同源性为99.58%,而推导的氨基酸序列仅存在1个氨基酸残基差异;DDAH2基因序列与普通牛比较相差1个碱基,序列同源性为99.91%,推导的氨基酸序列存在1个氨基酸残基差异。DDAH1和DDAH2基因在牦牛和犏牛睾丸组织中均有表达,DDAH1基因在犏牛睾丸组织中的表达量显著高于牦牛(P<0.05),是牦牛睾丸组织中表达量的1.5倍;DDAH2基因在犏牛睾丸中的表达量与牦牛相比差异不显著。说明DDAH基因表达在犏牛睾丸中上调可能与其雄性不育有关。

To determine the sequences of DDAH1 and DDAH2 genes and compare their expressions in the testes of yaks and sterile cattle - yaks, exploring the relationship between the genes and male sterility of cattle - yaks. Total RNA was extracted from yak testes and the cDNA sequences of the DDAH1 and DDAH2 genes in yaks were cloned by RT - PCR technique and sequencing analysis. The DDAH1 and DDAH2 ex- pressions in testes of yaks and cattle - yaks were detected using real - time fluorescent quantitative RT - PCR technique. The results showed that the sequences of DDAH1 and DDAH2 genes were 959 bp and 1 091 bp in length, respectively,both containing a coding sequence （CDS） region of 858 bp. The sequence of yak DDAH1 gene had four base differences compared with Bos tauras, and its sequence homology was 99.58%. However, there was only an amino acid residue difference in the corresponding deduced amino acid sequences. The sequence homolo- gy of yak DDAH2 gene was 99.91% , and there was a base difference compared with Bos tauras, and there was only an amino acid residue difference in the corresponding deduced amino acid sequences. The DDAH1 and DDAH2 genes were expressed in the testes of both yaks and cattle- yaks, and the DDAH1 expression level in the testis tissues of cattle- yaks was significantly higher （P 〈 0.05 ） than that in the yaks, which was 1.5 times of the expression level in the testis tissues of yaks, whereas there was no significant difference in the DDAH2 expression between the testes of cattle - yaks and yaks. The results indicate that the upregulated exprssion level of the DDAH gene in the testes of cattle - yaks may be associated with the male sterility of cattle - yaks.