犏牛PPP1R11基因的克隆及在睾丸中的表达规律研究

doi:10.11843/j.issn.0366-6964.2022.02.015

摘要/Abstract

摘要： 旨在克隆犏牛蛋白磷酸酶1调节亚基11（protein phosphatase 1 regulatory subunit 11，PPP1R11）基因，分析其在不同发育阶段睾丸中的表达与定位，为解析其在雄性生殖中的功能机制提供理论依据。本研究采集成年犏牛睾丸、附睾、心、肝、脾、肺、肾、大肠、小肠、胃、肌肉和脂肪组织（n=3），犏牛和牦牛在胎牛时期（5~6月龄）、幼年时期（1~2岁）和成年时期（3~4岁）睾丸（n=3）作为试验材料。通过RT-PCR技术克隆犏牛PPP1R11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）构建PPP1R11基因在犏牛不同组织中的表达谱，比较分析PPP1R11在犏牛和牦牛不同时期睾丸中的表达，利用免疫组织化学（immunohistochemistry，IHC）技术检测PPP1R11蛋白的细胞定位和表达。结果显示，犏牛PPP1R11基因CDS区为324 bp，编码107个氨基酸，犏牛PPP1R11蛋白与其他哺乳动物同源性较高；PPP1R11可与PPP1R2、PPP1R7、PPP1CB和UTP20等蛋白相互作用，互作蛋白功能主要与蛋白质的磷酸化相关，参与调控睾丸发育、精子发生和性成熟等生物学过程。PPP1R11在犏牛组织中广泛表达，其中在睾丸组织中的表达量最高，相对表达水平极显著高于其它组织（P<0.01）；该基因在犏牛睾丸中的表达量随年龄增长呈上升趋势，幼年和成年时期犏牛睾丸中PPP1R11的表达与同时期牦牛相比差异极显著（P<0.01）；IHC染色结果发现，PPP1R11蛋白在犏牛精原细胞和支持细胞中低表达并与牦牛存在显著差异，雄性犏牛初级精母细胞数量减少并且减数分裂阻滞于此。本研究表明，犏牛与牦牛PPP1R11在睾丸中存在时空表达差异，提示该基因可能与犏牛雄性不育相关，其具体作用机制有待进一步研究。

关键词: 犏牛, PPP1R11, 基因克隆, 基因表达, 减数分裂, 睾丸发育

Abstract: This study was conducted to clone the PPP1R11 gene in cattle-yak, detect the expression and location in testes of cattle-yak at different developmental stages, as to provide a theoretical basis for further research of the mechanism of function in male reproduction. The testis, epididymis, heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, muscle and adipose tissue of adult cattle-yak were collected (n=3), testicles of cattle-yak and yak in the fetal period (5-6 months old), juvenile period (1-2 years old) and adult period (3-4 years old) (n=3) were collected as experimental materials. The PPP1R11 CDS region of cattle-yak was cloned by RT-PCR and analyzed by bioinformatics softwares. qRT-PCR was used to detect the expression patterns of PPP1R11 mRNA in different tissues of cattle-yak, and compared the expression difference in testes at different development stages between cattle-yak and yak. The cell location and expression of PPP1R11 protein were detected by immunohistochemistry (IHC) staining. The results showed that the CDS region of PPP1R11 gene in cattle-yak was 324 bp, which encoded 107 amino acids. The corresponding PPP1R11 protein of cattle-yak had high homology with other mammals. The protein function prediction showed that PPP1R11 could interact with PPP1R2, PPP1R7, PPP1CB, UTP20 and other proteins, and mainly related to the phosphorylation of proteins, which could regulate biological processes such as testicular development, spermatogenesis and sexual maturity. PPP1R11 gene was wildly expressed in various tissues of cattle-yak, and the relative expression level in testis was significantly higher than other tissues (P<0.01). The expression of PPP1R11 gene in cattle-yak testis increased with age. Furthermore, there was significant difference of the expression level of PPP1R11 (P<0.01) in testis at juvenile and adult stage of cattle-yak compared with the counterpart of yak. In addition, IHC staining result showed that PPP1R11 protein was significantly lower expressed in cattle-yak spermatogonia and sertoli cells, and there was significant difference compared with yak, and the number of primary spermatocytes in cattle-yak was reduced significantly and meiosis arrested at this stage. This study showed that there were temporal and spatial differences in the expression of PPP1R11 between cattle-yak and yak testis, suggesting that PPP1R11 may be related to to male cattle-yak infertility. However, its specific mechanism needs to be further studied.

Key words: cattle-yak, PPP1R11, gene cloning, gene expression, meiosis, testis development

中图分类号:

S823.85

闵星宇, 杨丽雪, 于海玲, 胡宇磊, 杨满珍, 杨璐瑜, 李键, 熊显荣. 犏牛PPP1R11基因的克隆及在睾丸中的表达规律研究[J]. 畜牧兽医学报, 2022, 53(2): 481-492.

MIN Xingyu, YANG Lixue, YU Hailing, HU Yulei, YANG Manzhen, YANG Luyu, LI Jian, XIONG Xianrong. Cloning and Expression Analysis of Cattle-yak PPP1R11 Gene in Testis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 481-492.

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