Wang Pei, Zhang Xia, Huo Hailong, Wang Shuyan, Song Xue, Huo Jinlong
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan, China.
Teaching Affairs Department, Yunnan Vocational and Technical College of Agriculture, Kunming, Yunnan, China.
Anim Reprod. 2021 Feb 16;17(4):e20200521. doi: 10.1590/1984-3143-AR2020-0521.
Septin14 is an important spermatogenesis related gene involved in the pathogenesis of male infertility that has not been well studied. Here, full-length Septin14 cDNA of the Banna mini-pig inbred line (BMI) was cloned using the RACE method and expressed in pig kidney epithelial cells (PK15) and Rosetta (DE3) cells. Septin14 expression was identified in somatic tissues and testis in different developmental stages. The pig Septin14 CDS is 1,299 bp long, and encodes a peptide (or protein) of 432 amino acids (MW=50.4 kDa). Phylogenetic analysis indicated that pig Septin14 was highly evolutionarily conserved. Subcellular localization of GFP-tagged Septin14 fusion protein revealed that Septin14 was distributed throughout the testicular cells. Among 34 pig tissues, Septin14 mRNA was found specifically in testis and seminal vesicle. In six different postnatal developmental stages, the testicular level of Septin14 mRNA was barely detectable on day 2, while the highest level occurred on day 75. The spatiotemporal expression profile of Septin14, reported herein for the first time in pig, indicated that Septin14 might be involved in the division, development and apoptosis of germ cells. Furthermore, using a pET prokaryotic expression system, we expressed and isolated recombinant 67.9 kDa Septin14 protein.
Septin14是一个与精子发生相关的重要基因,参与男性不育症的发病机制,但目前尚未得到充分研究。在此,我们采用RACE方法克隆了版纳微型猪近交系(BMI)的全长Septin14 cDNA,并在猪肾上皮细胞(PK15)和Rosetta(DE3)细胞中进行表达。在不同发育阶段的体细胞组织和睾丸中均鉴定到了Septin14的表达。猪Septin14的CDS区长1299 bp,编码一个由432个氨基酸组成的肽(或蛋白质)(分子量=50.4 kDa)。系统发育分析表明,猪Septin14在进化上高度保守。绿色荧光蛋白标记的Septin14融合蛋白的亚细胞定位显示,Septin14分布于整个睾丸细胞中。在34种猪组织中,仅在睾丸和精囊中发现了Septin14 mRNA。在出生后的六个不同发育阶段,Septin14 mRNA在睾丸中的水平在第2天几乎检测不到,而在第75天达到最高水平。本文首次报道了猪Septin14的时空表达谱,表明Septin14可能参与生殖细胞的分裂、发育和凋亡。此外,我们利用pET原核表达系统表达并分离出了重组的67.9 kDa的Septin14蛋白。