Cloning and optimizing the expression of the DHDPS gene in the Medicago truncatula

Plant material and chemical preparation
 
Medicago truncatula seeds were grown and developed according to the method of Ellen et al., (2013) with slight modifications. The quality seeds were washed under running tap water and sterilized with 5 ml of concentrated sulphuric acid solution under the sterile laminar flow and washed thoroughly with sterile distilled water. Finally, the seeds were transferred to a petri dish and 5 ml of sterile distilled water were added with a pipette. The petri dish was covered by paraffin and kept it in a culture room (23°C; 16-h/8-h light/dark cycle at 30mmol m^-2 s^-1). Germinating seedlings were transferred to a hydrophonic system. After that, they were transferred to grow under natural environmental conditions. Almost  all the plants were developed well in the hydroponic system, using leaf material from three individual plants which were employed to constitute one biological replicate.
 
DNA isolation and PCR amplification of the DHDPS gene
 
Genomic DNA of M. truncatula was extracted from three weeks old leaves of M. truncatula via the improved CTAB (cetyltrimethyl ammonium bromide) method (Yan et al., 2018) and used as template in PCR amplification. Primer-BLAST was used to design primers and the specific primers DHDPSF: 5'-CACCATGAATGTTAGGAAATCGATTGACG-3' and DHDPSR: 5'-ATACCGACCAACTATGATAAAGTCTTC-3'), which was designed base on the coding DNA sequence (CDS) for the DHDPS gene (accession number on Genbank XM_013589555) was used for PCR. The concentrations of DNA were quantified by measuring absorbance using a NanoDrop (ND-1000) at 260-280 nm. To enable directional cloning further, the forward primers contain the CACC sequence (adapter) at the 5' end. These four nucleotides form a base pair with the GTGG overhang sequence in pET200/D-TOPO vector (Loc et al., 2013). For PCR amplification, two μl of DNA extracted genome (20ng/ml) was amplified in a 25 μl reaction mixture containing 1μM of each primer (10 pmol/ml), 12.5 μl of Master mix (Fermentas, Canada) (2.4 mM each deoxyribon- ucleotide) (dNTP) and 0.3 units Taq DNA polymerase (Fermentas, Canada) in the thermocycler (iCycler, BioRad). The thermocycling profile was as follow: initial denaturation at 95°C for 5 minutes; 30 repeated cycles of 95°C for 1 minute, 45°C for 1 minute and 72°C for 1 minute and a final extension of 72°C for 10 minutes and the end at 4°C.
       
The PCR product was electrophoresed with 1% agarose gel at 80 volts in 1×, TAE buffer and stained with ethidium bromide (0.5 μg/L) for 15 minutes. The stained gel was photographed under UV light using the Gel Documentation system (Bio-Rad).
 
Cloning and sequencing of DHDPS gene
 
The PCR product was eluted  from 1% agarose gel and purified by KIT Isolate II PCR and Gel (Bioline, USA) and it was inserted into pGEM®-T Easy vector (Promega, USA) according to the TA cloning method. The ligation component consisted of 50 ng pGEM®-T Easy vector (50 ng/µl), 5 µl buffer, 1µl T4 DNA ligase (3 unit/µl) and 3 µl PCR product (15 ng/µl). Distilled water was added to a make up a final volume of 10 µl. The ligation was incubated at 25°C for 1 hour and at 4°C overnight, and then ligation products were transformed into E. coli TOP10 cells (Invitrogen, USA) by the heat-shock method. The presence of the insert was determined by colony direct PCR followed by 1% agarose gel electrophoresis. Positive colonies (white colonies) were cultured on 5 ml of LB medium of 1% peptone, 1% NaCl, 0.5% yeast extract, 1.5% agar, pH: 7.0) supplemented with 50 µg/ml ampicillin, 100 mM Isopropylthio-β-galactoside (IPTG)  and 20 mg/ml X-Gal for biomass production. Recombinant pGEM®-T Easy vector was then isolated by EZ-10 Spin Column Plasmid DNA MiniPreps Kit, BS614 (Bio-Base INC). The nucleotide sequence of DHDPS gene was analysed by the method of dideoxy terminator and they were compared with nucleotide sequences from the GenBank database using the BLAST program from www.ncbi.nlm.nih.gov/BLAST.
 
Expression of DHDPS2 protein
 
The PCR product from recombinant pGEM®-T Easy vector was eleuted  from 1% agarose gel and purified by KIT Isolate II PCR and Gel (Bioline, USA), they were then ligated to a pET200/D-TOPO expression vector harboring T7 promoter (Invitrogen). The ligation component consisted of 20 ng pET200/D -TOPO vector, 1 ml salt solution and 9.6 ng PCR product. Distilled water was added to a final volume of 6 μl. The ligation was incubated at 25°C for 60 min. Recombinant pET200/D - TOPO vector was then transformed into E. coli StarTM BL21 (DE3) cells (Invitrogen) according to the manufacturer’s instructions. Expression of recombinant DHDPS2 protein in transformed E. coli StarTM BL21 (DE3) was first performed at 37°C on YJ medium culture of 2% glycerol, 1.5% tryptone, 2% yeast extract, 0.25% K₂HPO₄.12H₂O, 0.016% KH₂PO₄, 0.05% NaCl and 0.025% MgSO₄.7H₂O (Yang et al., 2008), supplemented with 1% glucose and 100 μg/ml kanamycin. The culture was carried out on a rotation shaker with a speed of 200 rpm to an OD600 of 0.8. Isopropyl b-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM for induction. Next assay for optimizing the expression of recombinant DHDPS2 protein in transformed E. coli StarTM BL21 (DE3), the effect of induction time after 2h, 4h, 6h, 8h, 10h and 12h, as well as the  concentration of IPTG for induction, were investigated varies from 0.2 to 1.5 mM, respectively.
               
After optimizing the time and concentration of IPTG induction, the different medium cultures were investigated with (1) LB medium: 0.5% yeast extract, 1% peptone and 1% NaCl and 1.5% agar (Vulfson et al., 2001), (2) SOB medium culture: 2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂ and 10 mM MgSO₄ (Shenet_al2007), (3) SOC medium culture: 2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄ and 20 mM glucose (Shen et al., 2007), (4) HSG medium culture: 1.49% glycerol, 0.7% yeast extract, 1.35% tryptone, 0.014% MgSO₄.H₂O, 0.15% KH₂PO₄, 0.23% K₂HPO₄ and 0.5% (Miksch et al., 2008) and (6) TB medium culture: 1.2% peptone, 2.4% yeast extract, 72mM K₂HPO₄, 17mM KH₂PO₄ and 0.4% glycerol (Shen et al., 2007) supplemented with 1% glucose and 100 μg/ml ampicillin. The culture was carried out on a rotation shaker with a speed of 200 rpm to an OD600 of 0.8. The cell biomass from each above medium culture assay was harvested by centrifugation at 15000 rpm/4°C for 1 min. The freezing and thawing at 42°C were performed with three repeats to break cells and total soluble protein was obtained by extraction according to Champion^TM pET 200 Directional TOPO^® expression kit (Invitrogen, USA). The expression level of 6xHis-DHDPS was assayed by electrophoresis  on 15% (w/v) polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) at 80V. The gel was then stained with Coomassie Blue R-250 and image was analysed by Quality One software (ver 4.1, BioRad).

Cloning and sequencing of the DHDPS gene
 
The PCR amplification of the mature peptide coding sequence of DHDPS gene from genomic DNA of M. truncatula leaf has been shown in Fig 1A. The PCR product had a band with a length of  922 bp detected using bioinformatics software. PCR product was purified and inserted into pGEM®-T Easy vector of  E. coli cells. The presence of the insert in transformed bacterial cells were determined by PCR amplification with M13 primers of vector and the results is in Fig 1B. DNA bands as shown in Fig 1B. It  was ~1123 bp in length (923 bp of insert and 200 bp of primer regions on vector).
 

       
Fig 2 display the sequencing profile of the DHDPS gene (accession no: XM_01358955.2) with 100% homology with DHDPS gene. had about 922 nucleotides. The BLAST search showed that the nucleotide and the amino acid sequences of DHDPS gene from M. truncatula leaf was 100% homology with M. truncatula 4-hydroxy-tetra-hydrodipicolinate synthase 2 (DHDPS2) (accession number: XM_013589555 from nucleotide 117 to 1037), coding for a long polypeptide of 307 amino acid with molecular mass of about 33495 Da from NCBI (Protein ID: XP_013445009.1) (Fig 3).
 

 

 
Expression of DHDPS2 gene
 
Expression of recombinant DHDPS2 polypeptides in E. coli. BL21 (DE3) cells on YJ culture medium with 0.5 mM IPTG induction, after analyzed through SDS-PAGE has been shown in Fig 4. The protein bands of three repeats transformed  E. coli cells with 0.5 mM IPTG induction after 5h (lane 1, 2 and 3) from SDS-PAGE gel as shown in Fig 4 revealed that the expression of recombinant DHDPS polypeptides in E. coli. BL21 (DE3) cells on YJ culture medium was expected to produce proteins of 37.2 kDa (33.5 kDa DHDPS subunit (Fig  3) and 3.7 kDa fusion fragment of the pET200/D-TOTO vector, (Loc et al., 2015). For the transformed E. coli cells without IPTG induction, a weak protein band of expected sizes were observed (Fig  4, NC), suggesting that induction with IPTG is necessary for optimizing the expression of DHDPS2 subunit gene.
 

 
The previous study reported that  the expression vector with a T7 lac promoter, final IPTG induce time and concentration should be optimized because of its great contribution to recombinant protein expression and serious harm to cell growth (Loc et al., 2013). In this research, after adding  0.5 mM IPTG into the medium, the target protein started to be synthesized and induction time was necessary for recombinant protein production. The optimal induction time for expression was examined by analyzing samples after 2h, 4h, 6h, 8h, 10 and 12h induction. The results showed that highest expression of DHDPS subunit occurred after 8h of induction (Fig 5). The effect of different concentrations IPTG on the DHDPS production was investigated. IPTG concentrations of 0.2 mM, 0.5 mM, 0.7 mM, 1.0 mM, 1.2 mM and 1.5 mM was used to induce expression of DHDPS subunits after 8h, and the result was shown in Fig 5. Comparison of the intensity of the bands in SDS-PAGE showed that 0.2mM IPTG is suitable enough for higher expression of DHDPS subunit (Fig 6).
 

 

       
The effect of six different culture mediums of LB, TB, HSG, YJ, SOB, SOC with 0.5 mM IPTG induction on the expression of recombinant DHDPS2 protein in E. coli. BL21 (DE3) cells have been shown in Fig 7. In fact, LB is one of the normal culture media to use for determining the expression of the recombinant general protein in E. coli. BL21 (DE3) cells (Loc et al., 2015, Ellen et al., 2013, Miksch et al., 2008). LB culture medium was also applied to detect the expression of recombinant general protein in E. coli. BL21 (DE3) cells in this assay and the result on SDS-PAGE gel showed a very weak band of molecular weight of about 37.2 kDa (Fig 5, LB) in comparing with other culture media under the same assay condition with 0.5 mM IPTG induction after 5h (see material and method). Among six different investigated culture mediums, the expression of recombinant DHDPS protein in E. coli. BL21 (DE3) cells on the TB, HSG and YJ culture mediums shown rather stronger band than SOB and SOC culture mediums (Fig 5).
 

       
Taking into account the results obtained from this study, it is possible to suggest that the highest expression of recombinant which designed to base on the coding DNA sequence (CDS) for the mature peptide of DHDPS protein in E. coli. BL21 (DE3) cells occurred on the HSG culture mediums after 8h of induction with 0.2 mM IPTG (BioRad).