Plant specific rna kit

To investigate PtDef transcription in different tissues, total RNA was isolated from mature leaves, young leaves, petioles, roots, and the upper and lower regions of stems of P. trichocarpa. P. trichocarpa seedlings treated with 200 mM NaCl, 200 μm abscisic acid (ABA), 200 μm SA, 200 μm JA, and 2 mM H₂O₂ were sampled at 0, 2, 4, 6, 8, 12, 24, and 48 h, and those subjected to 4 °C cold stress and 10% PEG 6000 were collected after 1, 2, 3, 4, 5, 6, and 7 days. In addition, a group of P. trichocarpa seedlings were wounded with S. populiperda and sampled at 0, 1, 3, 6, 9, 12, 24, 48, and 72 h. Following these treatments, leaves were immediately subjected to RNA extraction using a plant-specific RNA kit (Invitrogen). The housekeeping gene actin (XM_006370951.1) was used as an internal control for each reaction^{41 (link)–44 (link)}. Triplicate measurements were used to determine the mean values for each parameter.

Total RNA was extracted using a plant-specific RNA kit (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from RNA isolated from P. trichocarpa using M-MLV reverse transcriptase. Primers (Supplement Table 1) were designed and synthesized for defensin ORF cloning, the PCR system includes 2 µL forward and reverse primers, 2.0 µL cDNA as template, 5.0 µL 10× PCR buffer (Mg²⁺ plus), 1 µL 10 mM dNTPs, 0.5 µL rTaq DNA polymerase (Takara, Japan) and the ddH₂O was to a constant volume up to 50 µL. Also, the PCR reaction was performed as follows: 95 °C for 10 min, 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s and, finally, 72 °C for 10 min. Also the PtDef PCR product was purified (Takara, Japan) and inserted into the PEASY-T3 plasmid (TransGen Biotech, China). Subsequently, the positive clones were sequenced using the M13 primers. The nucleotide sequence, deduced amino acid sequence, and ORF of defensin from P. trichocarpa were analyzed using the ExPASy online package (http://www.expasy.org/translate/). We then calculated the molecular weight (Mw) of defensin and predicted its isoelectric point (pI) using the ExPASy pI/Mw tool (http://www.expasy.org/tools/pi_tool.html).