# Overexpression of _PtDXS_ Enhances Stress Resistance in Poplars
## Materials and Methods
### Overexpression Plasmid Construction and Transformation  
The open reading frame (ORF) of _PtDXS_ was cloned into pGWB9 using gateway technology: First, the ORF of _PtDXS_ was cloned into the pDONRTM/Zeo entry vector using the BP clonase according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The method of electroporation was used to transform the recombinant plasmid pDONRTM/Zeo-PtDXS into _E. coli_ Top10 cells and the positive clones were selected by solid LB medium containing 30 \(\upmu\)g/mL zeocin.  
Second, using the LR clones (Invitrogen), recombinant pDONRTM/Zeo-_PtDXS_ was recombined with the pGWB9 vector, and the electroporation was used to transform the recombinant plasmid pGWB9-PtDXS into _E. coli_ DH5\(\alpha\) cells and the positive clones were selected by solid LB medium containing 50 \(\upmu\)g/mL Kanamycin. pGWB9-_PtDXS_ was introduced into _Agrobacteriumum_ EHA105, which was used to infect poplar leaves.  
Transgenic operations are carried out in the following steps: Resistant shoots were obtained on selective differentiation medium containing 200 mg/L cefotaxime and 50 mg/L kanamycin. Next, putative resistant shoots were cultured on selective shooting MS medium containing 200 mg/L cefotaxime and 25 mg/L kanamycin. Subsequently, the putative resistant shoots were cultured on half-strength MS medium containing 200 mg/L cefotaxime and 15 mg/L kanamycin. Finally, the plants were transferred to the greenhouse.  
To detect whether the _PtDXS_ gene was inserted into the poplar genome, PCR reaction was performed using 35S primer and PtDXS-R primer as the upstream primer and the downstream primer as well as the genomic DNA of wild-type and potential transgenic lines as templates, respectively. Also, the PtDXS-F primer and PtDXS-R primer as well as the genomic DNA of wild-type and potential transgenic lines as templates were used to carry out PCR reaction. The _kanamycin_ gene was indentified by PCR in twelve transgenic lines and the wild-type poplars. The copy number of transgenic lines was investigated by Southern blotting. The CTAB method was used to achieve 10 \(\upmu\)g of genomic DNA, then the genomic DNA was digested with EcoRI at 37 \({}^{\circ}\)C for 4 h. Subsequently, the digested genomic DNA was separated on a 0.8% agarose gel at 15 V, and was transferred to a Hybond N+nylon membrane. Finally, based on the manufacturer's instructions, blotting was performed (Roche, Basel, Switzerland). We perform the PCR to synthesize a digoxygenin (dig)-labeled _Kan_-tagged cDNA fragment (665 bp), and choose Kan-tagged cDNA fragment as a probe for Southern blotting.  
In addition, in order to identify whether the _PtDXS_ gene was expressed in the poplar genome, twelve transgenic lines were selected for real-time quantitative polymerase chain reaction (qPCR) analysis. Moreover, to identify whether the _PtDXS_ gene was translated in poplar, total proteins from both WT and transgenic poplars were extracted and analyzed by 12% SDS-PAGE. Furthermore, the _PtDXS_ gene linked to the PGWB9 vector by homologous recombination will be expressed in fusion with the 6\(\times\)His tag located on the PGWB9 vector. So, western blotting was used to determine whether PtDXS could be specifically recognized by rabbit antiserum against His-DXS expressed in transgenic poplars.  
After 1 month of subculture, shoot tips (3 cm high) were cut from wild-type and transgenic poplars and placed on medium containing 3% (\(w/v\)) PEG6000 or 100 mM NaCl under a 16 h light/8 h dark cycle at 23 \({}^{\circ}\)C. The survival rate was investigated after 1 week.