Hostname: page-component-78c5997874-mlc7c Total loading time: 0 Render date: 2024-11-12T19:46:05.408Z Has data issue: false hasContentIssue false
  • English
  • Français

Expression, purification and identification of gibberellin-induced cysteine-rich protein of Gymanadenia conopsea

Published online by Cambridge University Press:  29 January 2010

Liu Yuan ,
Meng Guo-Quan ,
Zhou Jian-Ping ,
Zhang Teng ,
Feng Juan  and
Ren Zheng-long
Liu Yuan
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Meng Guo-Quan
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Zhou Jian-Ping
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Zhang Teng
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Feng Juan*
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
Ren Zheng-long
Affiliation:
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
*
*Corresponding author. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Primers bearing restriction enzyme sites for EcoR I and Hind III were designed according to the known partial cDNA sequence for gibberellin-induced cysteine-rich protein and were then used to amplify the full-length open reading frame (ORF) and signal peptide-truncated fragment of the gcgasa gene. Two fragments with lengths 319 and 238 bp were obtained and were further cloned into plasmid pET-32(a). Following transformation into Escherichia coli BL21(DE3), the fusion proteins were observed to appear at ~26.0 and 25.2 kDa after induction from 1 mmol/l isopropyl-beta-D-thiogalactopyronoside (IPTG). The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) of an ultra-thin section revealed that the presence of signal peptide gave rise to the formation of an inclusion body located in the periplasmic space; however, the absence of signal peptide greatly enhanced the solubility of the target protein. The expressed soluble protein was further purified by Ni2+-NTA affinity chromatography and gel filtration methods.

Keywords

Gymanadenia conopseagibberellin-induced cysteine-rich proteinprokaryotic expression
Type
Research Papers
Information
Chinese Journal of Agricultural Biotechnology , Volume 6 , Issue 3 , December 2009 , pp. 271 - 276
Copyright
Copyright © China Agricultural University 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Aubert, D, Chevillard, M, Dorne, AM, Arlaud, G and Herzog, M (1998) Expression patterns of GASA genes in Arabidopsis thaliana: the GASA4 gene is up-regulated by gibberellins in meristematic regions. Plant Molecular Biology 36: 871–883.CrossRefGoogle ScholarPubMed
Ben-Nissan, G, Lee, JY, Borohov, A and Weiss, D (2004) GIP, a Petunia hybrida GA-induced cysteine-rich protein: a possible role in shoot elongation and transition to flowering. Plant Journal 37: 229–238.CrossRefGoogle ScholarPubMed
Berrocal-Lobo, M, Segura, A, Moreno, M, Lopez, G, Garcia-Olmedo, F and Molina, A (2002) Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiology 128: 951–961.CrossRefGoogle ScholarPubMed
Betton, JM and Hofnung, M (1996) Folding a mutant maltose-binding protein of Escherichia coli which forms inclusion bodies. Journal of Biological Chemistry 271: 8046–8052.CrossRefGoogle ScholarPubMed
de la Fuente, JI, Amaya, I, Castillejo, C, et al. (2006) The strawberry gene FaGAST affects plant growth through inhibition of cell elongation. Journal of Experimental Botany 57: 2401–2411.CrossRefGoogle ScholarPubMed
Furukawa, T, Sakaguchi, N and Shimada, H (2006) Two OsGASR genes, rice GAST homologue genes that are abundant in proliferating tissues, show different expressions patterns in developing panicles. Genes & Genetic Systems 81: 171–180.CrossRefGoogle ScholarPubMed
Herzog, M, Dorne, AM and Grellet, F (1995) GASA, a gibberellin-regulated gene family from Arabidopsis thaliana related to the tomato GAST1 gene. Plant Molecular Biology 27: 743–752.CrossRefGoogle Scholar
Kotilainen, M, Helariutta, Y, Mehto, M, et al. (1999) GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida. Plant Cell 11: 1093–1104.CrossRefGoogle ScholarPubMed
Medina-Escobar, N, Cardenas, J, Valpuesta, V, Munoz-Blanco, J and Caballero, JL (1997) Cloning and characterization of cDNAs from genes differentially expressed during the strawberry fruit ripening processes by a MAST–PCR–SBDS method. Analytical Biochemistry 248: 288–296.CrossRefGoogle ScholarPubMed
Messens, J and Collet, JF (2006) Pathways of disulfide bond formation in Escherichia Coli. International Journal of Biochemistry and Cell Biology 38: 1050–1062.CrossRefGoogle ScholarPubMed
Roxrud, I, Lid, SE, Fletcher, JC, Schmidt, ED and Opsahl-Sorteberg, HG (2007) GASA4, one of the 14 member Arabidopsis GASA family of small polypeptides, regulates flowering and seed development. Plant and Cell Physiology 48: 471–483.CrossRefGoogle ScholarPubMed
Schunmann, PHD, Smith, RC, Lang, V, Matthews, PR and Chandler, PM (1997) Expression of XET-related genes and its correlation to elongation in leaves of barley (Hordeum vulgare L.). Plant Cell and Environment 20: 1439–1450.CrossRefGoogle Scholar
Segura, A, Moreno, M, Madueno, F, Molina, A and Garcia-Olmedo, F (1999) Snakin-1, a peptide from potato that is active against plant pathogens. Molecular Plant–Microbe Interactions 12: 16–23.CrossRefGoogle ScholarPubMed
Taylor, BH and Scheuring, CF (1994) A molecular marker for lateral root initiation: the RSI-1 gene of tomato (Lycopersicon esculentum Mill.) is activated in early lateral root primordia. Molecular & General Genetics 243: 148–157.CrossRefGoogle ScholarPubMed
Wigoda, N, Ben-Nissan, G, Granot, D, Schwartz, A and Weiss, D (2006) The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity. Plant Journal 48: 796–805.CrossRefGoogle ScholarPubMed
Zhou, JP, Yang, ZJ, Bai, LL, et al. (2006) Construction of a full-length cDNA library of Gymanadenia conopsea by modified oligo-capping. Acta Botanica Boreali-Occidentalia Sinica 26: 1874–1877 (in Chinese with English abstract).Google Scholar