Late embryogenesis abundant (LEA) proteins are speculated to protect against water stress deficit in plants. An over expression system for mungbean late embryogenesis abundant protein, emv2 was constructed in a pET29a vector, designated pET-emv2 which is responsible for higher expression under the transcriptional/translational control of T7/lac promoter incorporated in the Escherichia coli BL21 (DE3).Induction protocol was optimized for pET recombinants harboring the target gene. Overexpressed EMV2 protein was purified to homogeneity and the protein profile monitored by SDS-PAGE

Manickam A.

Rajesh S.

Raveendran M.

Sivakumar P.

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Journal of Stress Physiology & Biochemistry, Vol. 4, No. 3, 2008, pp. 9-16    ISSN 1997-0838Original Text Copyright © 2008 by Rajesh, Raveendran , Sivakumar, Manickam. ORIGINAL ARTICLEOverexpression Analysis of emv2 gene coding for Late Embryogenesis Abundant Protein from Vigna radiata (Wilczek)Rajesh S.1, 2, Raveendran M.1, Sivakumar P.1, 3, Manickam A.1 1Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore-641003, India2National Research Centre for Soybean, Indore-452001, Madhya Pradesh, India3Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, MalaysiaReceived July 7, 2008Abstract - Late embryogenesis abundant (LEA) proteins are speculated to protect against water stress deficit in plants. An over expression system for mungbean late embryogenesis abundant protein, emv2 was constructed in a pET29a vector, designated pET-emv2 which is responsible for higher expression under the transcriptional/translational control of T7/lac promoter incorporated in the Escherichia coli BL21 (DE3).Induction protocol was optimized for pET recombinants harboring the target gene. Overexpressed EMV2 protein was purified to homogeneity and the protein profile monitored by SDS-PAGE.Key words:LEA protein, Overexpression, Vigna, desiccationJOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 4 No. 3 2008Overexpression Analysis of emv2 gene…ORIGINAL ARTICLEOverexpression Analysis of emv2 gene coding for Late Embryogenesis Abundant Protein from Vigna radiata (Wilczek)Rajesh S.1, 2, Raveendran M.1, Sivakumar P.1, 3, Manickam A.1 1Centre for Plant Molecular Biology, Tamil Nadu Agricultural University, Coimbatore-641003, India2National Research Centre for Soybean, Indore-452001, Madhya Pradesh, India3Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, MalaysiaReceived July 7, 2008Abstract -  Late embryogenesis abundant (LEA) proteins are speculated to protect against   water   stress   deficit   in   plants.   An   over   expression   system   for   mungbean   late embryogenesis abundant protein, emv2 was constructed in a pET29a vector, designated pET-emv2 which is responsible for higher expression under the transcriptional/translational control of T7/lac promoter incorporated in the Escherichia coli BL21 (DE3).Induction protocol was optimized for pET recombinants harboring the target gene. Overexpressed EMV2 protein was purified to homogeneity and the protein profile monitored by SDS-PAGE.Key words:LEA protein, Overexpression, Vigna, desiccationLate   embryogenesis   abundant   (LEA)   proteins have been found in a wide range of plant species in response to water deficit resulting from desiccation, cold and osmotic stress. LEA-type proteins fall into a number   of   families,   with   diverse   structures   and functions (Close 1996; Ingram and Bartels 1996; Thomashow 1998). It has been suggested that LEA-type proteins act as water-binding molecules, in ion sequestration and in macromolecule and membrane stabilization (i.e. chaperone-like activity; Dure 1993; Close 1996; Ingram and Bartels 1996; Thomashow 1998, 1999). Group1 LEA proteins posses’ 20-mer motif conserved regions in form of repeat regions rich in Glycine residues. In our earlier studies, we had reported the presence of LEA-type proteins in the embryonic axes of the developing seeds of mungbean (Vigna   radiata)   referred   to   as   EMV   proteins (Manickam and Carlier, 1980). These proteins are classified into Group 1 LEA protein based on motif analysis and its functions were predicted based on the secondary structures (Rajesh and Manickam, 2006). The   discovery   and   use   of   new   stress-tolerance-associated genes, as well as heterologous genes, to confer plant stress tolerance has been the subject of ongoing efforts to obtain tolerant plants. Recently, we reported sense expression of  emv2  in indica rice plants   that   shall   tolerate   limited   water   situations (Rajesh et al., 2008).Overexpression of a single LEA-type protein is not always sufficient to confer plant stress tolerance. Although some progress of physiological roles of LEA proteins has been made, it is difficult to see the protective effect of over-expression of a single LEA protein gene introduced in transgenic plant, due to expression of the endogenous lea gene families that existed in genomic DNA of the host plant cells. Liu and   Zheng,   2005   demonstrated   that   expression system   of  E.   coli  is   a   simple,   convenient,   and effective   model   to   determine   the   function   of   a heterogeneous protein under stress treatments. Some plant genes related with dehydration stresses have been isolated by functional expression screening in JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 4 No. 3 200811 10Rajesh et alE. coli system. Most of these proteins such as CCTa, XVSAP1,   BBC1,   LEA3,   HSP100   and   ubiquitin proteins are generally located in cytoplasm of the plant cells (Miyasaka et al., 2000 and Tanaka et al., 2001 and 2002). In the present study, as a preliminary investigation to asses the overexpression potential of emv2 in prokaryotic system E coli and its subsequent assessment   for   salt   tolerance   studies,   we   have constructed a pET based recombinant overexpression system with  emv2  gene, coding for a LEA protein from Vigna radiata and the protein expression profile was electrophoretically analyzed. MATERIALS AND METHODSMaterialsThe insert emv2 gene (NCBI GenBank accession numbers U31211, Fig.1) coding for a Group 1 Late Embryogenesis Abundant- type protein from mung bean (Vigna radiata) is a proprietary gene isolated from our laboratory. pET29a vector and host cells E coli BL21 (DE3) from Novagen Inc, Germany.  All other   chemical   reagents/fine   chemicals   otherwise mentioned   are   purchased   from   Sigma-Aldrich, Bangalore, India.MethodsConstruction   of   pET-emv2  overexpression systemThe   insert  emv2  was   prepared   by   restriction digestion of plant transformation vector pCEMV2 constructed in our laboratory. Restriction digestion was done as per standard procedures (Sambrook et al.,   1989).   Approximately,   200-250   ng   each   of plasmid DNA were digested with 1μl of  Bam  HI (10U/μl) and  Hind III (20U/μl) (Bangalore Genei, Bangalore, India) in an appropriate buffer at 37ºC for 1   h.   The   digested   products   were   fractionated   on agarose gel (Fig.2). Insert DNA was eluted from the agarose gel following phenol: chloroform method. The gel was visualized over UV transilluminator and the desired band was excised with a sterile scalpel and frozen at -70 оC for 1 h and macerated into pieces with blunt ended micropipette tip. TE saturated phenol; pH 8.0 was added to this, mixed and incubated at 65 оC for solubilising   gel   pieces.   The  mix   was   centrifuged, supernatant  saved  and ethanol precipitated.  Eluted DNA was used for subcloning into pET29a at the Bam H1 and  Hind III cloning/expression region (Fig. 3) under the transcriptional/translational control of T7/lac promoter. The vector, pET29a was prepared following similar procedure and restriction digested. emv2 was ligated into the vector following sticky end ligation using T4 DNA ligase. Fresh competent cells of E coli BL21 (DE3) was prepared by treating the cells with 100mM CaCl2  and the pET recombinants harboring emv2  gene  were transformed into highly effective BL21 (DE3) host cells by heat shock treatment at 42 оC for 90 sec. The transformants were selected using kanamycin, subcultured and used for overexpression analysis.Overexpression and Purification of the  pET-EMV2Recombinant  E. coli, BL21  (DE3) harbouring pET-emv2 (pET29a + emv2) or  pET29a vector alone were grown in LB broth containing kanamycin 50 mgml-1 at 37°C till OD600 = ~0.6. Isopropyl b-D-thio galactopyranoside (IPTG) was added to each flask at a final concentration of 1 mM followed by further cell growth at 37 °C for 6 h (OD600 = ~1.3). This was followed   by   pelleting   of   cells   by   centrifugation, washing in sterile distilled water, resuspension in 500 ml of 50 mM Tris-HCl, pH 7.0 and sonication in TE buffer containing 1 mM PMSF, until more than 90% of   the   cells   were   lysed.   Sonicated   cells   were centrifuged at 7000 x g for 15 min at 4°C. The pellet was suspended in the TE buffer containing 1.0% triton X-100 and washed twice in the same buffer. Finally, the pellet was dissolved in a small volume of sterile   double   distilled   water   and   protein   profile analyzed by SDS-PAGE. RESULTSConstruction of pET-emv2 overexpression systemInsert   cDNA  emv2  and   the   cloning   vector pET29a was prepared following standard protocols. Sticky end ligation of the insert emv2  and pET29a was performed using T4 DNA ligase. The construct was designated as pET-emv2. The pET recombinants and also the control, pET29a vector were transformed into competent cells of E. coli BL21 (DE3) by heat shock treatment and induced for overexpression.Overexpression analysis of pET-emv2A 625 base pair cDNA fragment, emv2 encoding the   Late   embryogenesis   abundant   protein   from mungbean (Vigna radiata) was subcloned into the expression   vector   pET   29a   as   described   above, making the construct pET-emv2.  E. coli BL21 (DE3) cells were transformed with this plasmid or pET29a as a control and induced with IPTG as described above. After induction, cells were lysed and the polypeptide profiles compared by SDS-PAGE. Purity of eluted fractions from recombinant E. coli strains monitored   by   SDS-PAGE   analysis   showed   a prominent band of ~ 12 kDa in case of pET-emv2, whereas there was no distinct band of that size in pET29a   control   (Fig.4).   Other   prominent   protein bands as visualized were similar to the control. JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 4 No. 3 2008Overexpression Analysis of emv2 gene…Fig. 1. Organization of emv2, a LEA protein coding gene from Vigna radiata 20-mer amino acid repeat motif at position 181-240 is presented in red. Fig. 2.  Preparation of insert by restriction digestionThe figure shows unrestricted plasmid pCEMV2 (lane 1), BamH1 and Hind III sequentially digest DNA (lane 2) and 1kb DNA ladder given in lane M. JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 4 No. 3 200812Rajesh et alFig. 3.  Physical map and cloning/expression region of pET29 a (+) vectorOver expression system constructed with  mungbean late embryogenesis abundant protein gene, emv2 with flanking Bam H1 and Hind III sites. Cloning position indicated as red down arrows.JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 4 No. 3 200813Overexpression Analysis of emv2 gene…Fig. 4. Expression of pET-emv2 in E. coli BL21 (DE3)Cultures of E. coli BL21 (DE3) containing plasmids were made and supernatants prepared as described under methods. The figure shows samples from host cells BL21 uninduced (lane 1), BL21 induced (lane 2), pET-emv2 uninduced (lane 3) and pET-emv2 induced 12kDa EMV2 protein (lane 4). Markers are given in lane M. Numbers give molecular masses in kilodaltons.DISCUSSIONLEA proteins and heat-shock proteins have been shown to be involved in protecting macromolecules, such   as   enzymes   and   lipids   (Shinozaki   and Yamaguchi-shinozaki, 2000 and Seki  et al., 2002). Although   several   studies   have   been   extensively carried   out   documenting   the   responses   of   LEA proteins   to   different   abiotic   as   well   as   biotic conditions, the true biochemical function of these loosely related proteins is yet to be fully understood. The pET expression system is the most powerful system and is choice for the cloning and expression of recombinant proteins in  E. coli  and the target genes   cloned   are   driven   under   the transcriptional/translational   control   of   strong bacteriophage T7 signals; expression is induced by providing a source of T7 RNA polymerase in the host cell  with an  advantage that when  fully induced, almost all of the cell’s resources are converted to target gene expression and the desired product can comprise more than 50% of the total cell protein within   a   few   hours   after   induction(pET Manual, 2003). A 625 bp mungbean emv2 gene, containing   a   20-mer   amino   acid   repeat   motif (GGQTRKQQLGSEGYHEMGRK)   at   positions 181-240   and   a   hydrophilic   plant   signature (GETVVPGGT) at position 94-128 was cloned in a pET29a vector system and expressed in E. coli, and has shown to retain intact by expressing the EMV protein of mass 12,000 Daltons at a relatively higher level than background. These signature repeats in the JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 4 No. 3 200814Rajesh et alLEA proteins of Vigna radiata are similar and share identity of about 30% to the GsiB stress protein from Bacillus subtilis  (Stacy and  Aalen, 1998) which is induced by glucose or phosphate starvation, oxygen limitation, heat, oxidation and salinity (Volker  et al., 1994).   This   remarkable   conservation   suggests   an important role of LEA proteins in stress adaptation.Considering the higher expression level of emv2 gene, this gene can be overexpressed in plant system either singly or in tandem with our already reported emv1 gene (NCBI GenBank Acc. Number U31210) which has two such 20-mer repeat motifs at regions 44-63 and 64-83 (Rajesh and Manickam, 2006), to produce   dehydration   tolerant   protein   at   higher expression   level.   The   principle   of   functional expression screening is that due to the expression of foreign   plant   genes   carried   by   plasmid   DNA  in prokaryote cells, the host E. coli cells acquire stress tolerance. Hence at the next level, this protein will be further   tested   for   its   salt   tolerance   capacity   by MALDI-TOF and Spot assay experiments. Liu and Zheng, 2005 has demonstrated expression of a 22-mer motif containing Group 3 LEA protein from soybean and has reported to directly contribute to increasing stress tolerance of the bacteria host cells, and   also   indicated   that   some   of   the   protective mechanisms might be common in prokaryote and eukaryote under stress conditions (Garray-Arrayo et al.,  2000). The transgenic approaches using LEA proteins,   so   far   described   were   on   the   basis   of insights   gained   from   the   physiological   and biochemical studies of stress tolerance in different crop plants. Interestingly, any results gained from transgenic   studies   by   overexpression   of   these dehydration tolerant LEA genes will be miraculous and   will   help   us   to   better   understand   the physiological   functions   of   different   stress-related genes. ACKNOWLEDGEMENTSS.Rajesh is grateful to the Council of Scientific and Industrial Research, New Delhi for grant of Research Fellowship. REFERENCESClose,   T.J.  1996.     Dehydrins:   Emergence   of   a biochemical   role  of   a  family  of   plant dehydration proteins. Physiol. Plant., 97: 795-803Dure, L. 1993.  Structural motifs in Lea proteins. In. Plant Responses to Cellular Dehydration during   Environmental   Stress.  Current Topics in Plant Physiology, Vol  10.  TJ Close, EA Bray, (eds). American Society of Plant   Physiologists,   Rockville,   MD,   pp 91-103Garay-Arroyo,   A.,   J.M   .Colmenero-Flores,   A Garciarrubio and AA Covarrubias. 2000. Highly hydrophilic proteins in prokaryotes and   eukaryotes   are   common   during conditions of water deficit. J Biol. Chem., 275: 5668–5674Ingram ,G and D. Bartels. 1996. The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol., 47: 377-403Lium Y and Y. Zheng. 2005. 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Overexpression Analysis of emv2 gene coding for Late Embryogenesis Abundant Protein from Vigna radiata (Wilczek)

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