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Biologia




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Volume 69, Issue 8

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Isolation of cDNA and upstream sequence of a gene encoding trehalose-6-phosphate synthase 1 from Beauveria bassiana and its functional identification in Pichia pastoris

Ling Xie
  • Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, 230036, People’s Republic of China
  • School of Life Sciences, Anqing Teachers College, Anqing, 246011, People’s Republic of China
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/ Hongmei Chen / Zhangxun Wang
  • Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, 230036, People’s Republic of China
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/ Bo Huang
  • Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, 230036, People’s Republic of China
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Published Online: 2014-08-19 | DOI: https://doi.org/10.2478/s11756-014-0407-3

Abstract

Trehalose is an important molecule in fungal cells that helps to protect against various environmental stresses. In most fungi, trehalose-6-phosphate synthase 1 (TPS1) catalyzes the synthesis of trehalose-6-phosphate, and is the key enzyme for biosynthesis of this sugar. In this study, the full-length Beauveria bassiana tps1 gene sequence was determined. Full-length Bbtps1 (1,906 bp) included a 1,563 bp open reading frame that contained a 55 bp intron located between deduced amino acids 104 and 105. Bioinformatics analysis predicted that the BbTPS1 protein comprised 520 residues with a calculated pI value of 5.63 and a molecular weight of 58.3 kDa. Using DNA walking experiments, we determined the 2,963 bp upstream sequence that included several typical promoter elements and putative transcription factor binding sites, such as TATA-box, GC-box, Oct-1, CRE-BP, CdxA, and GATA. Stress-response and heat-shock elements were also found in this upstream sequence. Recombinant BbTPS1 was expressed in Pichia pastoris GS115 in order to probe the function of Bbtps1. SDS-PAGE analysis showed that the expressed protein had a molecular weight of approximately 60 kDa as expected. Enzymatic activity measurements revealed specific TPS1 activity that peaked at 1.38 U/mL at 96 h. This work provides a basis for further functional investigation of the mechanism of trehalose anabolism in B. bassiana. It could also assist the construction of engineered B. bassiana strains with enhanced stress tolerance.

Keywords: Beauveria bassiana; trehalose-6-phosphate synthase 1; gene cloning; upstream sequence; Pichia pastoris

  • [1] Al-Bader N., Vanier G., Liu H., Gravelat F.N., Urb M., Hoareau C.M., Campoli P., Chabot J., Filler S.G. & Sheppard D.C. 2010. Role of trehalose biosynthesis in Aspergillus fumigatus development, stress response, and virulence. Infect. Immun. 78: 3007–3018. http://dx.doi.org/10.1128/IAI.00813-09Web of ScienceCrossrefGoogle Scholar

  • [2] Bale J.S., van Lenteren J.C. & Bigler F. 2008. Biological control and sustainable food production. Philos. Trans. R. Soc. Lond. B Biol. Sci. 363: 761–776. http://dx.doi.org/10.1098/rstb.2007.2182CrossrefGoogle Scholar

  • [3] Bandara A., Fraser S., Chambers P.J. & Stanley G.A. 2009. Trehalose promotes the survival of Saccharomyces cerevisiae during lethal ethanol stress, but does not influence growth under sublethal ethanol stress. FEMS Yeast Res. 9: 1208–1216. http://dx.doi.org/10.1111/j.1567-1364.2009.00569.xCrossrefGoogle Scholar

  • [4] Bell W., Klaassen P., Ohnacker M., Boller T., Herweijer M., Schoppink P., Van der Zee P. & Wiemken A. 1992. Characterization of the 56-kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIF1, a regulator of carbon catabolite inactivation. Eur. J. Biochem. 209: 951–959. http://dx.doi.org/10.1111/j.1432-1033.1992.tb17368.xGoogle Scholar

  • [5] Bendtsen J.D., Nielsen H., von Heijne G & Brunak S. 2004. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340: 783–795. http://dx.doi.org/10.1016/j.jmb.2004.05.028CrossrefGoogle Scholar

  • [6] Cao Y., Wang Y., Dai B., Wang B., Zhang H., Zhu Z., Xu Y., Jiang Y. & Zhang G. 2008. Trehalose is an important mediator of Cap1p oxidative stress response in Candida albicans. Biol. Pharm. Bull. 31: 421–425. Web of ScienceCrossrefGoogle Scholar

  • [7] Cardoso F.C., Pinho J.M.R., Azevedo V. & Oliveira S.C. 2006. Identification of a new Schistosoma mansoni membranebound protein through bioinformatic analysis. Genet. Mol. Res. 5: 609–618. Google Scholar

  • [8] Chaudhuri P., Basu A. & Ghosh A.K. 2008. Aggregation dependent enhancement of trehalose-6-phosphate synthase activity in Saccharomyces cerevisiae. Biochim. Biophys. Acta 1780: 289–297. http://dx.doi.org/10.1016/j.bbagen.2007.12.002CrossrefWeb of ScienceGoogle Scholar

  • [9] Chaudhuri P., Basu A., Sengupta S., Lahiri S., Dutta T. & Ghosh A.K. 2009. Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae. Biochim. Biophys. Acta 1790: 368–374. http://dx.doi.org/10.1016/j.bbagen.2009.03.008CrossrefWeb of ScienceGoogle Scholar

  • [10] Cui S.Y. & Xia Y.X. 2009. Isolation and characterization of the trehalose-6-phosphate synthase gene from Locusta migratoria manilensis. Insect Sci. 16: 287–295. http://dx.doi.org/10.1111/j.1744-7917.2009.01268.xWeb of ScienceCrossrefGoogle Scholar

  • [11] Doehlemann G., Berndt P. & Hahn M. 2006. Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea. Microbiology 152: 2625–2634. http://dx.doi.org/10.1099/mic.0.29044-0CrossrefGoogle Scholar

  • [12] Farenhorst M., Mouatcho J.C., Kikankie C.K., Brooke B.D., Hunt R.H., Thomas M.B., Koekemoer L.L., Knols B.G. & Coetzee M. 2009. Fungal infection counters insecticide resistance in African malaria mosquitoes. Proc. Natl. Acad. Sci. USA 106: 17443–17447. http://dx.doi.org/10.1073/pnas.0908530106CrossrefGoogle Scholar

  • [13] Fillinger S., Chaveroche M.K., van Dijck P., de Vries R., Ruijter G., Thevelein J. & d’Enfert C. 2001. Trehalose is required for the acquisition of tolerance to a variety of stresses in the filamentous fungus Aspergillus nidulans. Microbiology 147: 1851–1862. Google Scholar

  • [14] Ghikas D.V., Kouvelis V.N. & Typas M.A. 2010. Phylogenetic and biogeographic implications inferred by mitochondrial intergenic region analyses and ITS1-5.8S-ITS2 of the entomopathogenic fungi Beauveria bassiana and B. brongniartii. BMC Microbiol. 10: 174. http://dx.doi.org/10.1186/1471-2180-10-174Web of ScienceCrossrefGoogle Scholar

  • [15] Hernandez M.M., Martinez-Villar E., Peace C., Perez-Moreno I. & Marco V. 2012. Compatibility of the entomopathogenic fungus Beauveria bassiana with flufenoxuron and azadirachtin against Tetranychus urticae. Exp. Appl. Acarol. 58: 395–405. http://dx.doi.org/10.1007/s10493-012-9594-1CrossrefWeb of ScienceGoogle Scholar

  • [16] Honda Y., Tanaka M. & Honda S. 2010. Trehalose extends longevity in the nematode Caenorhabditis elegans. Aging Cell 9: 558–569. http://dx.doi.org/10.1111/j.1474-9726.2010.00582.xCrossrefWeb of ScienceGoogle Scholar

  • [17] Iturriaga G., Suarez R. & Nova-Franco B. 2009. Trehalose metabolism: from osmoprotection to signaling. Int. J. Mol. Sci. 10: 3793–3810. http://dx.doi.org/10.3390/ijms10093793Web of ScienceCrossrefGoogle Scholar

  • [18] Li G.Q. & Moriyama E.N. 2004. Vector NTI, a balanced all-inone sequence analysis suite. Brief. Bioinform. 5: 378–388. http://dx.doi.org/10.1093/bib/5.4.378CrossrefGoogle Scholar

  • [19] Li Z.Z., Huang B., Chen M.j., Wang B. & Fan M.Z. 2011. Studies on the genus Beauveria in molecular era. Mycosystema 30: 823–835. Google Scholar

  • [20] Lopez M., Tejera N.A., Iribarne C., Lluch C. & Herrera-Cervera J.A. 2008. Trehalose and trehalase in root nodules of Medicago truncatula and Phaseolus vulgaris in response to salt stress. Physiol. Plant. 134: 575–582. http://dx.doi.org/10.1111/j.1399-3054.2008.01162.xWeb of ScienceCrossrefGoogle Scholar

  • [21] Meera A., Rangarajan L. & Bhat S. 2009. Computational approach towards finding evolutionary distance and gene order using promoter sequences of central metabolic pathway. Interdiscip. Sci. 1: 128–132. http://dx.doi.org/10.1007/s12539-009-0017-3CrossrefWeb of ScienceGoogle Scholar

  • [22] Nielsen M., Lundegaard C., Lund O. & Petersen T.N. 2010. CPHmodels-3.0-remote homology modeling using structureguided sequence profiles. Nucleic Acids Res. 38: 576–581. http://dx.doi.org/10.1093/nar/gkq535Web of ScienceCrossrefGoogle Scholar

  • [23] Plasterer T.N. 2000. PROTEAN — protein sequence analysis and prediction. Mol. Biotechnol. 16: 117–125. http://dx.doi.org/10.1385/MB:16:2:117CrossrefGoogle Scholar

  • [24] Rassette M.S., Pierpont E.I., Wahl T. & Berres M. 2011. Use of Beauveria bassiana to control northern fowl mites (Ornithonyssus sylviarum) on roosters in an agricultural research facility. J. Am. Assoc. Lab. Anim. Sci. 50: 910–915. Google Scholar

  • [25] Sharma S.C. & Anand M.S. 2006. Role of selenium supplementation and heat stress on trehalose and glutathione content in Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 133: 1–7. http://dx.doi.org/10.1385/ABAB:133:1:1CrossrefGoogle Scholar

  • [26] Silva Z., Alarico S. & da Costa M. 2005. Trehalose biosynthesis in Thermus thermophilus RQ-1: biochemical properties of the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. Extremophiles 9: 29–36. http://dx.doi.org/10.1007/s00792-004-0421-4CrossrefGoogle Scholar

  • [27] Silva Z., Alarico S., Nobre A., Horlacher R., Marugg J., Boos W., Mingote A.I. & da Costa M.S. 2003. Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose. J. Bacteriol. 185: 5943–5952. http://dx.doi.org/10.1128/JB.185.20.5943-5952.2003CrossrefGoogle Scholar

  • [28] Sun M., Ren Q., Liu Z., Guan G., Gou H., Ma M., Li Y., Liu A., Yang J., Yin H. & Luo J. 2011. Beauveria bassiana: Synergistic effect with acaricides against the tick Hyalomma anatolicum anatolicum (Acari: Ixodidae). Exp. Parasitol. 128: 192–195. http://dx.doi.org/10.1016/j.exppara.2011.03.012Web of ScienceCrossrefGoogle Scholar

  • [29] Suzuki H., Pabst M.J. & Johnston R.B. 1985. Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxideproducing NADPH oxidase in membrane fractions of human neutrophils and monocytes. J. Biol. Chem. 260: 3635–3639. Google Scholar

  • [30] Swislocka R., Oleksinski E., Regulska E., Kalinowska M. & Lewandowski W. 2007. Structural characterization of alkali metal 3-nitrobenzoates. J. Mol. Sruct. 834: 380–388. http://dx.doi.org/10.1016/j.molstruc.2006.11.051CrossrefGoogle Scholar

  • [31] Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28: 2731–2739. http://dx.doi.org/10.1093/molbev/msr121Web of ScienceCrossrefGoogle Scholar

  • [32] Thorat L.J., Gaikwad S.M. & Nath B.B. 2012. Trehalose as an indicator of desiccation stress in Drosophila melanogaster larvae: a potential marker of anhydrobiosis. Biochem. Biophys. Res. Commun. 419: 638–642. http://dx.doi.org/10.1016/j.bbrc.2012.02.065Web of ScienceCrossrefGoogle Scholar

  • [33] Thurkathipana N. & Mikunthan G. 2008. Eco-friendly management of hadda beetle using Beauveria bassiana in Brinjal. Commun. Agric. Appl. Biol. Sci. 73: 597–602. Google Scholar

  • [34] Van Houtte H., Vandesteene L., Lopez-Galvis L., Lemmens L., Kissel E., Carpentier S., Feil R., Avonce N., Beeckman T., Lunn J.E. & Van Dijck P. 2013. Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in abscisic acid-induced stomatal closure. Plant Physiol. 161: 1158–1171. http://dx.doi.org/10.1104/pp.112.211391CrossrefGoogle Scholar

  • [35] Wang Z.L., Lu J.D. & Feng M.G. 2012. Primary roles of two dehydrogenases in the mannitol metabolism and multi-stress tolerance of entomopathogenic fungus Beauveria bassiana. Environ. Microbiol. 14: 2139–2150. http://dx.doi.org/10.1111/j.1462-2920.2011.02654.xCrossrefWeb of ScienceGoogle Scholar

  • [36] Yu A.Q., Jin X.K., Li S., Guo X.N., Wu M.H., Li W.W. & Wang Q. 2013. Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis. Dev. Comp. Immunol. 41: 723–727. http://dx.doi.org/10.1016/j.dci.2013.08.013CrossrefGoogle Scholar

  • [37] Zhang F., Wang Z.P., Chi Z., Raoufi Z., Abdollahi S. & Chi Z.M. 2013. The changes in Tps1 activity, trehalose content and expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17-1 grown at different temperatures. Extremophiles 17: 241–249. http://dx.doi.org/10.1007/s00792-013-0511-2CrossrefWeb of ScienceGoogle Scholar

  • [38] Zhang J., Wang J., Li F., Sun Y., Yang C. & Xiang J. 2012. A trehalose-6-phosphate synthase gene from Chinese shrimp, Fenneropenaeus chinensis. Mol. Biol. Rep. 39: 10219–10225. http://dx.doi.org/10.1007/s11033-012-1897-0Web of ScienceCrossrefGoogle Scholar

  • [39] Zhang Q. & Yan T. 2012. Correlation of intracellular trehalose concentration with desiccation resistance of soil Escherichia coli populations. Appl. Environ. Microbiol. 78: 7407–7413. http://dx.doi.org/10.1128/AEM.01904-12CrossrefWeb of ScienceGoogle Scholar

  • [40] Zhang Y.J., Zhao J.H., Fang W.G., Zhang J.Q., Luo Z.B., Zhang M., Fan Y.H. & Pei Y. 2009. Mitogen-activated protein kinase hog1 in the entomopathogenic fungus Beauveria bassiana regulates environmental stress responses and virulence to insects. Appl. Environ. Microbiol. 75: 3787–3795. http://dx.doi.org/10.1128/AEM.01913-08CrossrefWeb of ScienceGoogle Scholar

About the article

Published Online: 2014-08-19

Published in Print: 2014-08-01


Citation Information: Biologia, Volume 69, Issue 8, Pages 959–967, ISSN (Online) 1336-9563, DOI: https://doi.org/10.2478/s11756-014-0407-3.

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