Abstract
Heat shock cognate 70 (HSC70) is a class of highly conserved proteins which functions as a molecular chaperon, participates in tolerance processes, and is involved in protein folding, degradation, targeting, translocation, and protein complex remodeling. In this study, the mRNA expression level of the Haliotis diversicolor HSC70 (HdHSC70) gene was detected by quantitative real-time PCR in different tissues and under different stresses. The results showed that the HdHSC70 gene was ubiquitously expressed in seven selected tissues. The highest expression level was detected in gills (P < 0.05). The expression level of the HdHSC70 gene was significantly upregulated by thermal stress, hypoxia stress, Vibrio parahaemolyticus infection, and combined thermal and hypoxia stress. The upregulation occurred at the early stage of stress. These results indicated that the HdHSC70 is an important component in the immune system of H. diversicolor and is involved in the early stress response. Meanwhile, 5′-flanking region sequence (2013 bp) of the HdHSC70 gene was cloned; it contains a putative core promoter region, heat shock element, CpG, and transcription elements including NF-1, Sp1, Oct-1, interferon consensus sequence binding protein (ICSBP), etc. In HEK 293T cells, the 5′-flanking region sequence is able to drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. The promoter activity increased after high-temperature treatment, which may be the immediate reason why the expression of the HdHSC70 gene was significantly upregulated by thermal stress. After the ICSBP-binding site was mutated, we found the luciferase activity significantly reduced, which suggested that the ICSBP-binding site has a certain enhancement effect on the activity of the HdHSC70 promoter.
Similar content being viewed by others
References
Amin J, Mestril R, Schiller P, Dreano M, Voellmy R (1987) Organization of the Drosophila melanogaster hsp70 heat shock regulation unit. Mol Cell Biol 7(3):1055–1062
Bahar MH, Hegedus D, Soroka J, Coutu C, Bekkaoui D, Dosdall L (2013) Survival and Hsp70 gene expression in Plutella xylostella and its larval Parasitoid diadegma insulare varied between slowly ramping and abrupt extreme temperature regimes. PLoS One 8:e73901
Bansal M, Kumar A, Yella VR (2014) Role of DNA sequence based structural features of promoters in transcription initiation and gene expression. Curr Opin Struct Biol 25:77–85
Boorstein WR, Ziegelhoffer T, Craig EA (1994) Molecular evolution of the HSP70 multigene family. J Mol Evol 38:1–17
Brade AM, Ngo D, Szmitko P, Li PX, Liu FF, Klamut HJ (2000) Heat-directed gene targeting of adenoviral vectors to tumor cells. Cancer Gene Ther 7(12):1566–1574
Cai XH, Huang YT, Zhang X, Wang SH, Zhou ZH et al (2014) Cloning, characterization, hypoxia and heat shock response of hypoxia inducible factor-1 (HIF-1) from the small abalone Haliotis diversicolor. Gene 534:256–264
Cheng W, Hsiao IS, Hsu CH, Chen JC (2004) Change in water temperature on the immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus. Dis Aquat Org 17:235–243
Cheng W, Lei J, Fox CW, Johnston JS, Zhu-Salzman K (2015) Comparison of life history and genetic properties of cowpea bruchid strains and their response to hypoxia. J Insect Physiol 75:5–11
Choi BG, Hepat R, Kim Y (2014) RNA interference of a heat shock protein, Hsp70, loses its protection role in indirect chilling injury to the beet armyworm, Spodoptera exigua. Comp Biochem Physiol A Mol Integr Physiol 168:90
Chuang KH, Ho SH, Song YL (2007) Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp (Penaeus monodon). Gene 405:10–18
Daugaard M, Rohde M, Jäättelä M (2007) The heat shock protein 70 family: highly homologous proteins with overlapping and distinct functions. FEBS Lett 581:3702–3710
Deb R, Sajjanar B, Singh U, Kumar S, Brahmane MP, Singh R et al (2013) Promoter variants at AP2 box region of Hsp70.1 affect thermal stress response and milk production traits in Frieswal cross bred cattle. Gene 532:230–235
Fujino K, Arai K, Kudo M, Takahashi K, Uematsu T, Okamura S (1992) Estimating rate of gene-centromere recombination at some isozyme loci in Haliotis diversicolor and H. discus hannai. Nippon Suisan Gakkaishi 58(3):445–459
Gong WN, Wan FH, Xie BY, Guo JY, Zhou Y (2009) Cloning and sequence analysis of promoters in hsp90 and hsp17.66 genes from Ageratina adenophora. J Trop Subtrop Bot 5:006 (In Chinese)
Grünberg S, Hahn S (2013) Structural insights into transcription initiation by RNA polymerase II. Trends Biochem Sci 38:603–611
Gu J, Huang LX, Shen Y, Huang LH, Feng QL (2012) Hsp70 and small Hsps are the major heat shock protein members involved in midgut metamorphosis in the common cutworm, Spodoptera litura. Insect Mol Biol 21:535–543
Gueguen Y, Cadoret JP, Flament D, Barreauroumiguière C, Girardot AL, Garnier J et al (2003) Immune gene discovery by expressed sequence tags generated from hemocytes of the bacteria-challenged oyster, Crassostrea gigas. Gene 303:139–145
Huang Y, Cai X, Zou Z, Wang S, Wang G, Wang Y, Zhang Z (2013a) Molecular cloning, characterization and expression analysis of three heat shock responsive genes from Haliotis diversicolor. Fish Shellfish Immunol. 36:590–599
Huang XD, Liu WG, Wang Q, Zhao M, Wu SZ, Guan YY et al (2013b) Molecular characterization of interferon regulatory factor 2 (IRF-2) homolog in pearl oyster Pinctada fucata. Fish Shellfish Immunol 34(5):1279–1286
Hunt C, Morimoto RI (1985) Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc Natl Acad Sci U S A 82:6455–6459
Jiang X, Zhai H, Wang L, Luo L, Sappington TW, Zhang L (2012) Cloning of the heat shock protein 90 and 70 genes from the beet armyworm, Spodoptera exigua, and expression characteristics in relation to thermal stress and development. Cell Stress Chaperones 17:67–80
Karlin S, Brocchieri L (1998) Heat shock protein 70 family: multiple sequence comparisons, function, and evolution. J Mol Evol 47:565–577
Karn H, Ovsenek N, Heikkila JJ (2011) Examination of the DNA sequence-specific binding properties of heat shock transcription factor in Xenopus laevis embryos. Biochem Cell Biol 70:1006–1013
Ke CH, You WW (2011) Advances in genetics and breeding of Haliotis diversicolor. J Xiamen Univ 50:425–430 (In Chinese)
Ke CH, Tian Y, Zhou SQ, Li FX (2000) Preliminary studies on hybridization of three species of abalone. Mar Sci 24:39–40 (In Chinese)
Kim YM, Im JY, Han SH, Kang HS, Choi I (2000) IFN-gamma up-regulates IL-18 gene expression via IFN consensus sequence-binding protein and activator protein-1 elements in macrophages. J Immunol 165:3198–3205
Kuang Q, Li J, You L, Shi C, Ji C, Guo X et al (2015) Identification and characterization of NF-kappaB binding sites in human miR-1908 promoter. Biomed Pharmacother 74:158–163
Li D, Li G, Wang K, Liu X, Li W, Chen X et al (2012) Isolation and functional analysis of the promoter of the amphioxus Hsp70a gene. Gene 510:39
Li Y, Jing L, Li H, Wang X, Yan Z, Fan Y et al (2014) Construction of an HSP 70B′ promoter-driven heat-inducible vectors pHSPshTERT and its anti-proliferative effect in breast cancer MCF-7 cells. Chin J Cancer Biother 21:130–135 (In Chinese)
Li J, Zhang H, Zhang X, Yang S, Yan T, Song Z (2015) Molecular cloning and expression of two heat-shock protein genes (HSC70/HSP70) from Prenant’s schizothoracin (Schizothorax prenanti). Fish Physiol Biochem 41:573–585
Liu PC, Chen YC, Huang CY, Lee KK (2000) Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome. Lett Appl Microbiol 31:433
Liu XD, Yan YH, Wang ZY, Cai MY, Ke CH (2008) A preliminary study on tolerance to high temperature and low salinity of Haliotis diversicolor reeve. J Jimei Univ 13:301–303 (In Chinese)
Mahroof R, Zhu KY, Subramanyam B (2005) Changes in expression of heat shock proteins in Tribolium castaneum (Coleoptera: Tenebrionidae) in relation to developmental stages, exposure time, and temperature. Ann Entomol Soc Am 98:100–1077
Mukhopadhyay I, Nazir A, Saxena DK, Chowdhuri DK (2010) Heat shock response: hsp70 in environmental monitoring. J Biochem Mol Toxicol 17:249–254
Pei H, Hu H, Zhang X, Su C, Song X (2007) Cloning and functional analysis of the heat-inducible promoter at HSP70b. Chin Agric Sci Bull 23:82–86 (In Chinese)
Scheller M, Foerster J, Heyworth CM, Waring JF, Löhler J, Gilmore GL et al (1999) Altered development and cytokine responses of myeloid progenitors in the absence of transcription factor, interferon consensus sequence binding protein. Blood 94:3764–7371
Shao Y, Li C, Zhang W, Xu W, Duan X, Li Y et al (2016) Cloning and comparative analysis the proximal promoter activities of arginase and agmatinase genes in Apostichopus japonicus. Dev Comp Immunol 65:299–308
Shen Q, Zhao L, Xie G, Wei P, Yang M, Wang S et al (2016) Cloning three Harmonia axyridis (Coleoptera: Coccinellidae) heat shock protein 70 family genes: regulatory function related to heat and starvation stress. J Entomol Sci 50:168–185
Shi J, Fu M, Zhao C, Zhou F, Yang Q, Qiu L (2016) Characterization and function analysis of hsp60 and hsp10 under different acute stresses in black tiger shrimp, penaeus monodon. Cell Stress Chaperones 21(2):1–18
Shim JK, Jung DO, Park JW, Kim DW, Ha DM, Lee KY (2006) Molecular cloning of the heat-shock cognate 70 (Hsc70) gene from the two-spotted spider mite, Tetranychus urticae, and its expression in response to heat shock and starvation. Comp Biochem Physiol B Biochem Mol Biol 145:288–295
Shu Y, Du Y, Wang J (2011) Molecular characterization and expression patterns of Spodoptera litura heat shock protein 70/90, and their response to zinc stress. Comp Biochem Physiol A Mol Integr Physiol 158:102–110
Song X, Zhang H, Wang L, Zhao J, Mu C, Song L et al (2011) A galectin with quadruple-domain from bay scallop Argopecten irradians is involved in innate immune response. Dev Comp Immunol 35:592–602
Sun Y, Zhao J, Sheng Y, Xiao YF, Zhang YJ, Bai LX et al (2016a) Identification of heat shock cognate protein 70 gene (Alhsc70) of Apolygus lucorum and its expression in response to different temperature and pesticide stresses. Insect Sci 23:37–49
Sun Y, Xin Z, Wang G, Shi L, Zeng X, Wang Y, Zhang Z (2016b) PI3K-AKT signaling pathway is involved in hypoxia/thermal-induced immunosuppression of small abalone Haliotis diversicolor. Fish Shellfish Immunol 59:492–508
Tamura T, Ozato K (2002) ICSBP/IRF-8: its regulatory roles in the development of myeloid cells. J Interf Cytokine Res Off J Int Soc Interf Cytokine Res 22:145–152
Thanaphum S, Haymer D (1998) A member of the hsp70 gene familyfrom the Mediterranean fruit fly, Ceratitis capitata. Insect Mol Biol 7:63–72
Wang IM, Contursi C, Masumi A, Ma X, Trinchieri G, Ozato K (2000) An IFN-gamma-inducible transcription factor, IFN consensus sequence binding protein (ICSBP), stimulates IL-12 p40 expression in macrophages. J Immunol (Baltimore, Md: 1950) 165:271–279
Wang H, Li X, Bajić VB (2004) Neural-statistical model of tata-box motifs in eukaryotes. In: Bajiśc VB (ed) Bioinformatics and biocomputing. PhysicaMVerlag, New York, pp 123–156
Wang X, Xu C, Wang X, Wang D, Wang Q, Zhang B (2006) Heat shock response and mammal adaptation to high elevation (hypoxia). Sci China Ser C Life Sci 49:500–512 (In Chinese)
Wang H, Li K, Zhu JY, Fang Q, Ye GY, Wang H, et al (2012) Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp, pteromalus puparum in response to environmental stresses. Arch Insect Biochem Physiol 79(4-5):247–263
Wang Q, Sham KW, Ogawa S, Li S, Parhar IS, Cheng CH et al (2013a) Regulation of the two kiss promoters in goldfish (Carassius auratus) by estrogen via different ERα pathways. Mol Cell Endocrinol 375:130–139
Wang TT, Wang N, Liao XL, Meng L, Liu Y, Chen SL (2013b) Cloning, molecular characterization and expression analysis of heat shock cognate 70 (Hsc70) cDNA from turbot (Scophthalmus maximus). Fish Physiol Biochem 39:1377–1386
Yi SY, Sun AQ, Sun Y, Yang JY, Zhao CM, Liu J (2006) Differential regulation of Lehsp23.8 in tomato plants: analysis of a multiple stress-inducible promoter. Plant Sci 171:398
Yoshimi T, Odagiri K, Hiroshige Y, Yokobori S, Takahashi Y, Sugaya Y, Miura T (2009) Induction profile of HSP70-cognate genes by environmental pollutants in Chironomidae. Environ Toxicol Pharmacol 28:294–301
Zhang ZH (2010) Clone of heat shock protein 70 (HSP70) genes in Crassostrea hongkongensis and response of the HSC70 gene to pollutants. Dissertation. Jinan University (In Chinese)
Zhang XH, Qi YX, Wang YQ, Wang ZB (2010) Prediction and analysis of CpG island and TATA-box in fruit fly promotor region. China J Bioinforma 8:82–90 (In Chinese)
Zhang X, Huang Y, Cai X, Zou Z, Wang G, Wang S et al (2014) Identification and expression analysis of immune-related genes linked to Rel/NF-κB signaling pathway under stresses and bacterial challenge from the small abalone Haliotis diversicolor. Fish Shellfish Immunol 41:200–208
Zhao KK, Cui YG, Liu JY (2011) Research progress of heat shock protein 10. Med Recapitulate 1:1–3 (in Chinese)
Zhao C, Zhang X, Li F, Huan P, Xiang J (2013) Functional analysis of the promoter of the heat shock cognate 70 gene of the Pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol 34:397–401
Zhuang LF, Wei H, Lin JR, Zhong BX (2011) Identification of Bombyx mori hsp70 promoter and its function. Chin J Cell Biol 33:503–509 (In Chinese)
Funding
The work was supported by the Natural Science Foundation of China (No. 41176152, No. 41006105).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Li, Y., Zhang, T., Zhang, X. et al. Heat shock cognate 70 gene in Haliotis diversicolor: responses to pathogen infection and environmental stresses and its transcriptional regulation analysis. Cell Stress and Chaperones 23, 335–346 (2018). https://doi.org/10.1007/s12192-017-0842-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12192-017-0842-5