4.5. Isolation of C. echinata mRNA The RNeasy Plant Mini kit was used to isolate the mRNA. Briefly, C. echinata seeds, harvest tree or eight weeks after flowering were frozen in liquid nitrogen. 100 mg of the resulting powder was incubated, according to the manufacturer, in a buffer containing guanidinium thiocyanate and β- mercaptoethanol, and then purified into a QIAshredder column. 4.6. C. echinata cDNA synthesis and amplification by polymerase chain reaction (PCR) The cDNA synthesis was performed with the ImProm-IITM Reverse Transcriptase, total mRNA and the oligo primer race adapter 3 ′ as primer, as recommended by the manufacturer. The degenerate oligonucleotide ODN-CeEI (5 ′ TTY GTN GTN GAY ACN GAR GRN AAY YTN HTN CAR AAY GGN GG 3 ′), based on the N-terminal amino acid sequence of CeEI (Cruz-Silva et al., 2013), was used as forward primer and the Abridged Universal Amplification Primer - AUAP - (5 ′ GGC CAC GCG TCG ACT AGT AC 3 ′) was used as reverse primer. Briefly, cDNA (2.0 μg) were incubated with Taq polymerase (5 U), oligonucleotide AUAP (50 pM), degenerate oligonucleotide (50 pM), dNTP mix (0.20 mM) e MgCl 2 (2.5 mM), in a total volume of 50 μL. Reactions with only one oligonucleotide were also performed as control. It was used pre-denaturation (94 ◦ C for 5 min), PCR amplification of 35 cycles (denaturing at 94 ◦ C for 40 s, annealing at 68 ◦ C for 40 s, and extension at 72 ◦ C for 100 s), and final extension (72 ◦ C for 5 min). The amplified DNA fragments were analyzed by gel agarose (1%) electrophoresis containing 1.27 μM ethidium bromide. The DNA fragment of 700 bp, corresponding to the CeEI DNA fragment, was purified in agarose using QIAEX II gel extraction kit and ligated into pGEM-T easy cloning vector.

Published as part of Cruz-Silva, Ilana, Gozzo, Andrezza Justino, Nunes, Viviane Abreu, Tanaka, Aparecida Sadae & Araujo, Mariana da Silva, 2021, Bioengineering of an elastase inhibitor from Caesalpinia echinata (Brazil wood) seeds, pp. 1-10 in Phytochemistry (112595) (112595) 182 on page 6, DOI: 10.1016/j.phytochem.2020.112595, http://zenodo.org/record/8291489