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. 2019 May 20;9(12):6968-6985.
doi: 10.1002/ece3.5264. eCollection 2019 Jun.

Genomewide identification and analysis of heat-shock proteins 70/110 to reveal their potential functions in Chinese soft-shelled turtle Pelodiscus sinensis

Tengfei Liu  1 , Yawen Han  1 , Ye Liu  1 , Huiying Zhao  1
Affiliations

Genomewide identification and analysis of heat-shock proteins 70/110 to reveal their potential functions in Chinese soft-shelled turtle Pelodiscus sinensis

Tengfei Liu et al. Ecol Evol. .

Abstract

Heat-shock proteins 70/110 (Hsp70/110) are vital molecular chaperones and stress proteins whose expression and production are generally induced by extreme temperatures or external stresses. The Hsp70/110 family is largely conserved in diverse animals. Although many reports have studied and elaborated on the characteristics of Hsp70/110 in various species, the systematic identification and analysis of Hsp70/110 are still poor in turtles. In this study, a genomewide search was performed, and 18 candidate PsHSP70/110 family genes were identified in Chinese soft-shelled turtle, Pelodiscus sinensis. These PsHSP70/110 proteins contained the conserved "heat shock protein 70" domain. Phylogenetic analysis of PsHSP70/110 and their homologs revealed evolutionary conservation of Hsp70/110 across different species. Tissue-specific expression analysis showed that these PsHSP70/110 genes were differentially expressed in different tissues of P. sinensis. Furthermore, to examine the putative biological functions of PsHSP70/110, the dynamic expression of PsHSP70/110 genes was analyzed in the testis of P. sinensis during seasonal spermatogenesis following germ cell apoptosis. Notably, genes such as PsHSPA1B-L, PsHSPA2, and PsHSPA8 were significantly upregulated in P. sinensis testes along with a seasonal decrease in apoptosis. Protein interaction prediction revealed that PsHSPA1B-L, PsHSPA2, and PsHSPA8 may interact with each other and participate in the MAPK signaling pathway. Moreover, immunohistochemical analysis showed that PsHSPA1B-L, PsHSPA2, and PsHSPA8 protein expression was associated with seasonal temperature variation. The expression profiling and interaction relationships of the PsHSPA1B-L, PsHSPA2, and PsHSPA8 proteins implied their potential roles in inhibiting the apoptosis of germ cells in P. sinensis. These results provide insights into PsHSP70/110 functions and will serve as a rich resource for further investigation of HSP70/110 family genes in P. sinensis and other turtles.

Keywords: Chinese soft‐shelled turtle Pelodiscus sinensis; expression profiling; heat‐shock proteins 70/110; protein interaction; regulation of apoptosis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Characterization of the identified PsHSP70/110 genes in Pelodiscus sinensis. (a) Phylogenetic analysis of PsHSP70/110 proteins using bootstrap values with 1,000 replications. (b) A diagram of conserved domains in PsHSP70/110 proteins
Figure 2
Figure 2
Overview of Hsp70/110 proteins in 13 species. (a) The number of HSP70/110 genes in different species. MYA: million years ago. (b) Phylogenetic relationships and classification of HSP70/110 homologous proteins using bootstrap values with 1,000 replications. The green circle indicates the PsHSP70/110 proteins in Pelodiscus sinensis. Asterisks in different colors indicate the different subfamilies by their classification. Ac, Anolis carolinensis; Am, Alligator mississippiensis; Cm, Chelonia mydas; Cp, Chrysemys picta bellii; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mg, Meleagris gallopavo; Mm, Mus musculus; Oa, Ornithorhynchus anatinus; Tm, Terrapene mexicana triunguis; Xl, Xenopus laevis
Figure 3
Figure 3
Expression patterns of PsHSP70/110 genes in different tissues of Pelodiscus sinensis. Each bar shows the mean ± SEM of triplicate assays
Figure 4
Figure 4
Heat map of PsHSP70/110 gene expression in the testis of Pelodiscus sinensis based on RNA‐seq analysis
Figure 5
Figure 5
Dynamic expression of PsHSP70/110 genes in the testis of Pelodiscus sinensis in April (Spring), July (Summer), and October (Autumn) determined by qRT‐PCR analysis. A different letter with a value indicates a significant difference at p < 0.05. Each bar shows the mean ± SEM of triplicate assays
Figure 6
Figure 6
Interaction networks of PsHSP70/110 proteins. The catalysis, reaction, and binding relationships are indicated with purple, black, and blue lines, respectively. The green arrows represent the positive interaction relationship
Figure 7
Figure 7
Immunostaining of PsHSPA1B‐L, PsHSPA2, and PsHSPA8 in the testis of Pelodiscus sinensis during spermatogenesis. Arrowhead, Spermatogonia; arrow, spermatozoa; Lc, Leydig cell; Lu, lumen. Scale bar = 20 µm
Figure 8
Figure 8
Characterization of PsHSPA1B‐L, PsHSPA2, and PsHSPA8 proteins. (a) Comparison of the predicted 3D protein structures among PsHSPA1B‐L (green), PsHSPA2 (blue), and PsHSPA8 (red). (b–d) Phylogenetic analysis of PsHSPA1B‐L, PsHSPA2, PsHSPA8 (in green circles), and their homologs from other species using bootstrap values with 1,000 replications. Ac, Anolis carolinensis; Am, Alligator mississippiensis; Cm, Chelonia mydas; Cp, Chrysemys picta bellii; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mg, Meleagris gallopavo; Mm, Mus musculus; Oa, Ornithorhynchus anatinus; Tm, Terrapene mexicana triunguis; Xl, Xenopus laevis
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