Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China.
Faculty of Animal Science and Technology, Yunnan Agricultural University, Kunming, 650201, China; Institute of Sericultural and Apiculture, Yunnan Academy of Agricultural Sciences, Mengzi, 661101, China.
Comput Biol Chem. 2021 Jun;92:107474. doi: 10.1016/j.compbiolchem.2021.107474. Epub 2021 Mar 10.
Tree shrews are more closely related to primate animals than rodents in many aspects. In addition, they also possess several advantageous characteristics including small body size, high brain-to-body mass ratio, low cost of feeding and maintenance, short reproductive cycle and life span, which make them promising novel laboratory animals to replace more precious larger primate animals. Testis-specific serine/threonine kinase (Tssk) plays important roles in spermatogenesis and/or the regulation of sperm function. However, studies on Tssk in tree shrews have not been reported yet. In the present study, the full-length sequences of five members of the Tssk family in tree shrews were cloned and their CDS region sequences were analyzed by basic bioinformatics. The phylogenetic tree and prokaryotic protein expression system of Tssk gene of tree shrews were constructed. The mRNA expressions of Tssk genes in 11 tissues/organs from tree shrews were studied. The results showed that: 1. the length of the CDS region of tree shrew Tssk gene for Tssk1B, Tssk2, Tssk3 (variant X1 / X2), Tssk4 (variant X1 / X2) and Tssk6 is 1080bp, 1077bp, 867 / 807bp, 1014 / 984bp, 822bp, respectively, encoding 359, 358, 288/268, 337/327 and 273 amino acids, respectively; the cloned sequences of Tssk genes have been submitted to GenBank with the following accession numbers: KX091161(Tssk1B), KX091162(Tssk2), KX091163(Tssk3 variant X1)/KX091164(Tssk3 variant X2), KX091165(Tssk4variant X1)/KX091166(Tssk4variant X2), KX091160(Tssk6). 2. All tree shrew Tssk proteins distribute in cytoplasm, indicating that they are hydrophilic and non-secretory proteins, with multiple phosphorylation sites of serine and/or threonine. In addition, they are all mixed proteins with similar tertiary structures sharing a highly conserved functional domain of S_TKc (Serine/Threonine protein kinases, catalytic domain). 3.The molecular phylogenetic tree of five Tssk genes in tree shrews indicates that they are neither rodent nor primate animal, but are closely related to primate animals. 4. Five members of the Tssk recombinant proteins in tree shrews were successfully obtained using the constructed prokaryotic protein expression system. 5. Five Tssk genes are specifically expressed in the testis and/or sperm of tree shrews. Additionally, small amount of Tssk1B was expressed in several tissues other than testis and sperm. Limited mRNA levels of Tssk2 and Tssk4 were expressed in the brain, while mRNA of Tssk3 or Tssk6 could only be detected in the testis and sperm. This study will provide fundamental data on reproductive biology of tree shrews, which paves a way for further studying Tssk's biological function in this novel model animal.
树鼩在许多方面与灵长类动物的亲缘关系比啮齿类动物更密切。此外，它们还具有一些有利的特征，包括体型小、脑体比高、饲养和维持成本低、繁殖周期和寿命短，这使得它们成为有前途的新型实验动物，可以替代更珍贵的大型灵长类动物。睾丸特异性丝氨酸/苏氨酸激酶（Tssk）在精子发生和/或精子功能调节中发挥重要作用。然而，树鼩中 Tssk 的研究尚未报道。本研究克隆了树鼩 Tssk 家族的五个成员的全长序列，并通过基本生物信息学方法分析了它们的 CDS 区序列。构建了树鼩 Tssk 基因的系统发育树和原核蛋白表达系统。研究了树鼩 11 种组织/器官中 Tssk 基因的 mRNA 表达。结果表明：1. 树鼩 Tssk1B、Tssk2、Tssk3（X1/X2 变体）、Tssk4（X1/X2 变体）和 Tssk6 的 Tssk 基因 CDS 区长度分别为 1080bp、1077bp、867/807bp、1014/984bp 和 822bp，分别编码 359、358、288/268、337/327 和 273 个氨基酸；已将 Tssk 基因的克隆序列提交至 GenBank，登录号分别为：KX091161（Tssk1B）、KX091162（Tssk2）、KX091163（Tssk3 X1）/KX091164（Tssk3 X2）、KX091165（Tssk4 X1）/KX091166（Tssk4 X2）、KX091160（Tssk6）。2. 所有树鼩 Tssk 蛋白都分布在细胞质中，表明它们是亲水的非分泌蛋白，具有多个丝氨酸和/或苏氨酸的磷酸化位点。此外，它们都是混合蛋白，具有相似的三级结构，共享高度保守的 S_TKc（丝氨酸/苏氨酸蛋白激酶，催化结构域）功能域。3. 树鼩 5 个 Tssk 基因的分子系统发育树表明，它们既不是啮齿类动物，也不是灵长类动物，而是与灵长类动物密切相关。4. 利用构建的原核蛋白表达系统成功获得了树鼩 5 个 Tssk 重组蛋白。5. 5 个 Tssk 基因在树鼩睾丸和/或精子中特异性表达。此外，Tssk1B 少量表达于睾丸和精子以外的几种组织中。Tssk2 和 Tssk4 的 mRNA 水平在大脑中有限表达，而 Tssk3 或 Tssk6 的 mRNA 只能在睾丸和精子中检测到。本研究将为树鼩生殖生物学提供基础数据，为进一步研究这种新型模型动物中 Tssk 的生物学功能奠定基础。
