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Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs from Galeruca daurica (Coleoptera: Chrysomelidae) and their expression analysis

  • Y. Tan (a1), Y. Zhang (a1), Z.-J. Huo (a1), X.-R. Zhou (a1) and B.-P. Pang (a1)...


Galeruca daurica (Joannis) is a new outbreak pest in the Inner Mongolia grasslands in northern China. Heat shock protein 10 and 60 (Hsp10 and Hsp60) genes of G. daurica, designated as GdHsp10 and GdHsp60, were cloned by rapid amplification of cDNA ends techniques. Sequence analysis showed that GdHsp10 and GdHsp60 encoded polypeptides of 104 and 573 amino acids, respectively. Sequence alignment and phylogenetic analysis clearly revealed that the amino acids of GdHsp10 and GdHsp60 had high homology and were clustered with other Hsp10 and Hsp60 genes in insects which are highly relative with G. daurica based on morphologic taxonomy. The mRNA expression analysis by real-time PCR revealed that GdHsp10 and GdHsp60 were expressed at all development stages and in all tissues examined, but expressed highest in eggs and in adults’ abdomen; both heat and cold stresses could induce mRNA expression of GdHsp10 and GdHsp60 in the 2nd instar larvae; the two Hsp genes were expressed from high to low with the extension of treatment time in G. daurica eggs exposed to freezing point. Overall, our study provides useful information to understand temperature stress responses of Hsp60 and Hsp10 in G. daurica, and provides a basis to further study functions of Hsp60/Hsp10 relative to thermotolerance and cold hardiness mechanism.


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Boettinger, L., Oeljeklaus, S., Guiard, B., Rospert, S., Warscheid, B. & Becker, T. (2015) The mitochondrial heat shock protein 70 (Hsp70) and Hsp10 cooperate in the formation of Hsp60 complexes. Journal of Biological Chemistry 290, 1161111622.
Bose, S., Weikl, T., Bügl, H. & Buchner, J. (1996) Chaperone function of HSP90-associated proteins. Science 274, 17151717.
Bozaykut, P., Kartal, O.N. & Karademir, B. (2014) Regulation of protein turnover by heat shock proteins. Free Radical Biology Medicine 77, 195209.
Cappello, F., de Macario, E.C., Marasà, L., Zummo, G. & Macario, A.J. (2008) Hsp60 expression, new locations, functions, and perspectives for cancer diagnosis and therapy. Cancer Biology & Therapy 7, 801809.
Chow, A.M., Ferriere-Pages, C., Khalouei, S., Reynaud, S. & Brown, I.R. (2009) Increased light intensity induces heat shock protein Hsp60 in coral species. Cell Stress & Chaperones 14, 469476.
Clare, D.K. & Saibil, H.R. (2013) ATP-driven molecular chaperone machines. Biopolymers 99, 846859.
Corrao, S., Campanella, C., Anzalone, R., Farina, F., Zummo, G., Conway, M.E., Macario, A.J., Cappello, F. & Rocca, G. (2010) Human Hsp10 and early pregnancy factor (EPF) and their relationship and involvement in cancer and immunity: current knowledge and perspectives. Life Science 86, 145152.
Cui, Y.D., Du, Y.Z., Lu, M.X. & Qiang, C.K. (2010) Cloning of the heat shock protein 60 gene from the stem borer, Chilo suppressalis, and analysis of expression characteristics under heat stress. Journal of Insect Science 10, 100.
Denlinger, D.L. (2002) Regulation of diapause. Annual Review of Entomology, 47, 93122.
Dunn, S.R., Pernice, M., Green, K., Hoegh-Guldberg, O. & Dove, S.G. (2012) Thermal stress promotes host mitochondrial degradation in symbiotic cnidarians: are the batteries of the reef going to run out? PLoS ONE 7(7), e39024.
Fang, L.S., Huang, S.P. & Lin, K.L. (1997) High temperature induces the synthesis of heat-shock proteins and the elevation of intracellular calcium in the coral Acropora grandis. Coral Reefs 16, 127131.
Feder, M.E. & Hofmann, G.E. (1999) Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annual Review of Physiology 61, 243282.
Gao, J.C., Zhou, X.R., Pang, B.P., Bao, X., Luo, J.P. & Erdengqimuge, (2015) Effects of low temperature on the survivorship and development of overwintering eggs of Galeruca daurica (Coleoptera: Chrysomelidae). Acta Entomologica Sinica 58(8), 881886.
Ge, L.Q., Huang, L.J., Yang, G.Q., Song, Q.S., Stanley, D., Gurr, G.M. & Wu, J.C. (2013) Molecular basis for insecticide-enhanced thermo tolerance in the brown planthopper Nilaparvata lugens Stal (Hemiptera: Delphacidae). Molecular Ecology 22, 56245634.
Georgopoulos, C. & Welch, W. (1993) Role of the major heat shock proteins as molecular chaperones. Annual Review of Cell Biology 9(1), 601634.
Ghosh, J.C., Dohi, T., Kang, B.H. & Altieri, D.C. (2008) Hsp60 regulation of tumor cell apoptosis. Journal of Biological Chemistry 283, 51885194.
Gupta, R.S. (1995) Evolution of the chaperonin families (HSP60, HSP10 and TCP-1) of proteins and the origin of eukaryotic cells. Molecular Microbiology 15(1), 111.
Hayer-Hartl, M., Bracher, A. & Hartl, F.U. (2016) The GroEL-GroES chaperonin machine: a nano-cage for protein folding. Trends in Biochemical Sciences 41(1), 6276.
Huang, L.H. & Kang, L. (2007) Cloning and inter specific altered expression of heat shock protein genes in two leaf miner species in response to thermal stress. Insect Molecular Biology 16, 491500.
Kastle, M. & Grune, T. (2012) Interactions of the proteasomal system with chaperones: protein triage and protein quality control. Progress in Molecular Biology and Translational Science 109, 113160.
Kayukawa, T. & Ishikawa, Y. (2009) Chaperonin contributes to cold hardiness of the onion aggot Delia antique through repression of depolymerization of actin at low temperatures. PLoS ONE 4(12), e8277.
Kayukawa, T., Chen, B., Miyazaki, S., Itoyama, K., Shinoda, T. & Ishikawa, Y. (2005) Expression of mRNA for the t-complex polypeptide-1, a subunit of chaperonin CCT, is up-regulated in association with increased cold hardiness in Delia antiqua. Cell Stress & Chaperones 10, 204210.
King, A.M. & MacRae, T.H. (2015) Insect heat shock proteins during stress and diapause. Annual Review of Entomology 60, 5975.
Krebs, R.A. & Feder, M.E. (1997) Deleterious consequences of Hsp70 over-expression in Drosophila melanogaster larvae. Cell Stress & Chaperones 2(1), 6071.
Krebs, R.A. & Feder, M.E. (1998) Hsp70 and larval thermo tolerance in Drosophila melanogaster: how much is enough and when is more too much? Journal of Insect Physiology 44(11), 10911101.
Kultz, D. (2005) Molecular basis of the cellular stress response. Annual Review of Physiology 67, 225257.
Li, H., Zhou, X.R., Pang, B.P. & Chang, J. (2014) Supercooling capacity and cold hardiness of Galeruca daurica (Coleoptera: Chrysomelidae). Acta Entomologia Sinica 57(2), 212217.
Li, Z.W., Li, X., Yu, Q.Y., Xiang, Z.H., Kishino, H. & Zhang, Z. (2009) The small heat shock protein (sHSP) genes in the silkworm, Bombyx mori, and comparative analysis with other insect sHSP genes. BMC Evolutionary Biology 9, 215.
Livak, K.J. & Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25, 402408.
Meng, X., Liu, P., Li, J., Gao, B.Q. & Chen, P. (2014) Physiological responses of swimming crab Portunus trituberculatus under cold acclimation: antioxidant defense and heat shock proteins. Aquaculture 434, 1117.
Meyer, E., Aglyamova, G.V. & Matz, M.V. (2011) Profiling gene expression responses of coral larvae (Acropora millepora) to elevated temperature and settlement inducers using a novel RNA-Seq procedure. Molecular Ecology 20, 35993616.
Michaud, M.B., Teets, N.M., Peyton, J.T., Blobner, B.M. & Denlinger, D.L. (2011) Heat shock response to hypoxia and its attenuation during recovery in the flesh fly, Sarcophaga crassipalpis. Journal of Insect Physiology 57, 203210.
Michaud, S., Marin, R. & Tanguay, R.M. (1997) Regulation of heat shock gene induction and expression during Drosophila development. Cellular and Molecular Life Science 53(1), 104113.
Neupert, W. (1997) Protein import into mitochondria. Annual Review of Biochemistry 66, 863917.
Olsen, K., Ritson, W.R., Ochrietor, J.D., Paul, V.J. & Ross, C. (2013) Detecting hyperthermal stress in larvae of the hermatypic coral Porites astreoides: the suitability of using biomarkers of oxidative stress versus heat-shock protein transcriptional expression. Marine Biology 160, 26092618.
Pfaffl, M.W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29, e45.
Prinsloo, E., Setati, M.M., Longshaw, V.M. & Blatch, G.L. (2009) Chaperoning stem cells: a role for heat shock proteins in the modulation of stem cell self-renewal and differentiation? BioEssays 31(4), 370377.
Punyavathi, S., Bhat, M.A. & Manjunatha, H.B. (2017) Comparative proteome analysis and thermal stress induced changes in the embryo of poly- and bi-voltine strains of Bombyx mori. Journal of Applied Biology & Biotechnology 5(2), 059067.
Rinehart, J.P., Li, A., Yocum, G.D., Robich, R.M., Hayward, S.A.L. & Denlinger, D.L. (2007) Up-regulation of heat shock proteins is essential for cold survival during insect diapause. Proceedings of the National Academy of Sciences 104, 1113011137.
Rodriguez-Lanetty, M., Harii, S. & Hoegh-Guldberg, O. (2009) Early molecular responses of coral larvae to hyperthermal stress. Molecular Ecology 18, 51015114.
Rosic, N.N., Kaniewska, P., Chan, C.K.K., Ling, E.Y.S., Edwards, D., Dove, S. & HoeghGuldberg, O. (2014) Early transcriptional changes in the reef-building coral Acropora aspera in response to thermal and nutrient stress. BMC Genomics 15, 1052.
Roth, M.S., Goericke, R. & Deheyn, D.D. (2012) Cold induces acute stress but heat is ultimately more deleterious for the reef-building coral Acropora yongei. Scientific Reports 2, 240.
Saibil, H.R. & Ranson, N.A. (2002) The chaperonin folding machine. Trends in Biochemical Science 27, 627632.
Sasibhushan, S., Ponnuvel, K.M. & Vijayaprakash, N.B. (2012) Diapause specific gene expression in the eggs of multivoltine silkworm, Bombyx mori, identified by suppressive subtractive hybridization. Comparative Biochemistry and Physiology B 161, 371379.
Seveso, D., Montano, S., Strona, G., Orlandi, I., Galli, P. & Vai, M. (2016) Hsp60 expression profiles in the reef-building coral Seriatopora caliendrum subjected to heat and cold shock regimes. Marine Environmental Research 19, 111.
Sharmas, S., Rohilla, M.S. & Tiwari, P.K. (2007) Developmental and hyperthermia-induced expression of the heat shock proteins HSP60 and HSP70 in tissues of the housefly Musca domestica: an in vitro study. Genetics & Molecular Biology 30(1), 159168.
Shi, J.X., Fu, M.J., Zhao, C., Zhou, F.L., Yang, Q.B. & Qiu, L.H. (2016) Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, Penaeus monodon. Cell Stress and Chaperones 21, 295312.
Sokolova, I.M. & Portner, H.O. (2001) Temperature effects on key metabolic enzymes in Littorina saxatilis and L. obtusata from different latitudes and shore levels. Marine Biology 139(1), 113126.
Tammariello, S.P., Rinehart, J.P. & Denlinger, D.L. (1999) Desiccation elicits heat shock protein transcription in the flesh fly, Sarcophaga crassipalpis, but does not enhance tolerance to high or low temperatures. Journal of Insect Physiology 45, 933938.
Tan, Y., Zhou, X.R. & Pang, P. (2017) Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis). Bulletin of Entomology Research 107(3), 359368.
Wang, H., Li, K., Zhu, J.Y., Fang, Q. & Ye, G.Y. (2012) Cloning and expression of heat shock protein genes from the endoparasitoid wasp, Pteromalus puparum in response to environmental stresses. Archives of Insect Biochemistry and Physiology 79, 247263.
Wang, H.S., Wang, X.H., Zhou, C.S., Huang, L.H., Zhang, S.F., Guo, W. & Kang, L. (2007) cDNA cloning of heat shock proteins and their expression in the two phases of the migratory locust. Insect Molecular Biology 16, 207219.
Xu, D.X., Sun, L., Liu, S.L., Zhang, L.B., Ru, X.S., Zhao, Y. & Yang, H.S. (2014) Molecular cloning of heat shock protein 10 (Hsp10) and 60 (Hsp60) cDNAs and their expression analysis under thermal stress in the sea cucumber Apostichopus japonicus. Comparative Biochemistry and Physiology B 171, 4957.
Xu, Z., Horwich, A.L. & Sigler, P.B. (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388, 741750.
Zhang, G., Storey, J.M. & Storey, K.B. (2011) Chaperone proteins and winter survival by a freeze tolerant insect. Journal of Insect Physiology 57, 11151122.
Zhang, L.J., Wang, K.F., Jing, Y.P., Zhuang, H.M. & Wu, G. (2015) Identification of heat shock protein genes hsp70s and hsc70 and their associated mRNA expression under heat stress in insecticide-resistant and susceptible diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). European Journal of Entomology 112(2), 215226.
Zhou, X.R., Gao, J.C. & Pang, B.P. (2016) Effects of temperature on the termination of Egg diapause and post-diapause embryonic development of Galeruca daurica (Coleoptera: Chrysomelidae). Environmental Entomology 45(4), 10761080.



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