犏牛和牦牛ESCO2基因的序列特征及其在睾丸中的表达对比分析

旨在克隆犏牛和牦牛姐妹染色单体内聚建立蛋白2（establishment of sister chromatid cohesion N-acetyltransferase 2，ESCO2）基因，并分析其在不同发育阶段睾丸中的表达与定位，为进一步解析ESCO2在减数分裂过程中的作用机制提供理论依据。本研究以健康雄性犏牛及牦牛为试验动物，根据年龄分为胎牛组（5~6月龄）、幼年组（1~2岁）和成年组（3~4岁），每组各3头。通过RT-PCR技术克隆犏牛和牦牛ESCO2基因并进行生物信息学分析，采用实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）检测ESCO2基因在犏牛不同组织中的表达谱，比较分析ESCO2在犏牛和牦牛不同时期睾丸中的表达规律，利用免疫组织化学（immunohistochemistry，IHC）技术检测ESCO2蛋白的细胞定位和表达差异。结果显示，犏牛ESCO2基因（GenBank登录号：MW198470） CDS区为1 833 bp，编码610个氨基酸，与牦牛相比，犏牛ESCO2序列第301~319位多19个氨基酸，另有3个氨基酸突变；犏牛ESCO2蛋白序列与黄牛的同源性高于其他哺乳动物；ESCO2可能与SMC3、SMC1A、PDS5A、PDS5B、STAG2等蛋白相互作用，互作蛋白功能与姐妹染色单体凝聚、减数分裂细胞周期、DNA修复、细胞分裂和染色体重构等生物学过程相关。ESCO2在犏牛各组织中均有表达，但在睾丸中的相对表达水平显著高于其它组织（P<0.05）；在犏牛睾丸中的表达随年龄增长呈上升趋势，幼年和成年时期犏牛睾丸中ESCO2的表达显著低于同时期牦牛（P<0.05）；IHC染色结果发现，雄性犏牛减数分裂阻滞于初级精母细胞，ESCO2蛋白在犏牛初级精母细胞中无表达并与牦牛存在差异。本研究结果表明，犏牛与牦牛的ESCO2基因、蛋白序列差异较大，且在睾丸发育过程中的表达模式差异显著，这可能是引起雄性犏牛减数分裂阻滞及不育的原因之一，其具体作用机制有待进一步研究。

Sequence Characteristics and Comparative Expression Analysis in Testis of ESCO2 Gene in Cattle-yak and Yak

The aim of this study was to clone the establishment of sister chromatid cohesion N-acetyltransferase 2(ESCO2) gene, analyze its expression and localization in the testis at different developmental stages in cattle-yak and yak, and provide a theoretical basis for further study the role mechanism of ESCO2 in the male meiosis process. Healthy male cattle-yaks and yaks were used as experimental animals in this study, they were divided into fetal cattle group (5-6 months old), juvenile group (1-2 years old) and adult group (3-4 years old), with 3 animals in each group. The CDS region of ESCO2 gene of cattle-yak and yak were cloned by RT-PCR and its sequence characteristics were analyzed by bioinformatics softwares. qRT-PCR was used to detect the expression patterns of ESCO2 gene in different tissues of cattle-yak. The expression of ESCO2 mRNA in testes at different developmental stages of cattle-yak and yak were detected by qRT-PCR, the differences of cell location and expression of ESCO2 protein were detected by immunohistochemistry (IHC) staining. The results showed that the CDS region of ESCO2 genes in cattle-yak was 1 833 bp(MW198470), it encoded 610 amino acids. Compared with yak, the ESCO2 protein sequence of cattle-yak had more 19 amino acids at position 301-319 aa and there were 3 amino acid mutations. The cattle-yak and cattle had higher homology than other mammals based on the protein sequence of ESCO2. Interaction network showed that ESCO2 mainly interacted with SMC3, SMC1A, PDS5A, PDS5B, STAG2 and these proteins were related to sister chromatid condensation, meiotic cell cycle, DNA repair, cell division and chromosome remodeling. ESCO2 was expressed in all tissues of the cattle-yak, but its relative expression level in the testis was significantly higher than those in other tissues (P<0.05). In addition, the spatial expression of ESCO2 in testes of cattle-yak showed an upward trend with age, and the relative expression abundance of ESCO2 in cattle-yak at juvenile and adult stages was significantly lower than that of yak(P<0.05). IHC staining showed that meiosis of male cattle-yak was blocked in primary spermatocytes and ESCO2 protein was differentially expressed in primary spermatocytes between cattle-yak and yak. The research suggested that there was obvious difference in the sequence and expression pattern of ESCO2 between cattle-yak and yak, which may be one of reasons caused male sterility in cattle-yak, but the specific mechanism need further study.