Identification of a heat shock cognate protein 70 gene in Chinese soft-shell turtle (Pelodiscus sinensis) and its expression profiles under thermal stress

Abstract

Abstract:The heat shock cognate protein 70 (Hsc70) is a member of a 70-kDa heat shock protein (HSP70) family that functions as molecular chaperones. In this study, a novel Hsc70 gene from Chinese soft-shelled turtle (Pelodiscus sinensis) (tHsc70) was identified. The tHsc70 full-length complementary DNA (cDNA) is 2 272 bp long with a 1 941-bp open reading frame (ORF) encoding 646 amino acids. Three characteristic signature regions of the HSP70 family, two major domains of an adenosine triphosphate (ATP)/guanosine triphosphate (GTP) binding domain (ABD), and a substrate-binding domain (SBD) were present in the predicted tHsc70 amino acid sequence. The tHsc70 gene was expressed in Escherichia coli BL21 and the expression product reacted with the anti-Hsc70 mouse monoclonal antibody by Western blotting. Homology analysis revealed that tHsc70 shared identity from 53.9% to 87.7% at the nucleotide level, and 49.1% to 99.5% at the amino acid level with the known Hsc70s. Phylogenetic analysis showed that tHsc70 was clustered together with the Hsc70 gene of another reptile species (Alligator mississippiensis). The tHsc70 was expressed in the liver, lung, heart, and skeletal muscle. The expression patterns of tHsc70 messenger RNA (mRNA) differed among different tissues under different durations of heat stress at 40 °C. Adaptation at 25 °C for 1 h after heat stress was also different among tissues and length of heat stress. Irrespective of different profiles of expression under heat stress, tHsc70 may play roles in protecting turtles from thermal stress.

Loggerhead sea turtle embryos (Caretta caretta) regulate expression of stress response and developmental genes when exposed to a biologically realistic heat stress

“…Wide-scale upregulation of HSP70, HSP90 and sHSP families in response to thermal stress is common in a number of fish (Fangue et al 2006;Liu et al 2013;Newton et al 2013;Smith et al 2013), insects (see King & MacRae 2015) and urchins (Runcie et al 2012), including during embryogenesis (Krone et al 1997;Runcie et al 2012). The observed significant increase in expression of hspa8 (aka hsc70) under thermal stress has been suggested to result in higher thermotolerance in fish (Fangue et al 2006) and is similar to observations in the Chinese soft-shelled turtle Pelodiscus sinensis (Li et al 2012). This upregulation of hspa8 might suggest that the study population at Dirk Hartog Island may be at least somewhat adapted to elevated temperatures during development; however, further research should compare geographically distinct populations to determine the relative thermotolerance of this population.…”

“…) and is similar to observations in the Chinese soft‐shelled turtle Pelodiscus sinensis (Li et al . ). This upregulation of hspa8 might suggest that the study population at Dirk Hartog Island may be at least somewhat adapted to elevated temperatures during development; however, further research should compare geographically distinct populations to determine the relative thermotolerance of this population.…”

Prevalence and antimicrobial susceptibility ofAeromonasspecies from diseased Chinese soft-shelled turtles (Trionyx sinens)

“…Chinese soft‐shelled turtle ( Trionyx sinensis ) is one of the most important commercially cultured aquaculture species in Asian countries with high nutritional and medicinal values (Li, Kang, Zhang, Zhu & Fang ; Ip, Lee, Wong & Chew ). Its annual yield in Zhejiang province, China, has reached 155 000 tons accounting for 46% of the total yield in China (Chen et al .…”

Identification and Functional Analysis of Interleukin-1β in the Chinese Soft-Shelled Turtle Pelodiscus sinensis

“…Truncated tIL-1β sequence-encoding amino acids 118–276 (predicted as 19 kDa mature peptide) were amplified using the primer set rtIL-1-F and -R. The fragment was then cloned into pET32a(+) and verified by sequencing. The recombinant tIL-1β was expressed using the prokaryotic expression system and purified with Ni-IDA agarose (Weisi Co. Ltd., Beijing, China) as previously described [ 30 ]. The size and purity of the protein was confirmed by SDS-PAGE and Western blotting using anti-His(6)-Tag antibody (Sigma, St. Louis, MO, USA).…”

“…All qPCR primer sets were verified as having amplification efficiency of 95%–105% and their amplification specificities were confirmed by analyzing the melting curve as a single peak with each primer set. The reaction conditions were slightly adjusted as previously described [ 30 ]. Fold changes of the gene transcripts were calculated as 2 - ΔΔCt [ 31 ].…”