Computational protocol: Overexpression of BdMATE Gene Improves Aluminum Tolerance in Setaria viridis

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Protocol publication

[…] Searches for the Sorghum bicolor MATE (SbMATE) gene in the Phytozome database were performed in order to find orthologs in the genome of B. distachyon. Based on the different sequences found, the alignment of these sequences was generated using the Clustal Omega (). Analysis of conserved protein domains and the prediction of the molecular and cellular functions of these domains were evaluated by the online softwares FFPred and MEMSAT (; ). The phylogenetic tree was generated based on amino acid sequences of major grasses () that have been studied with MATE gene, using the Geneious software () for alignments and MEGA7 software () for building phylogenetic tree. [...] After 7-days-old plantlets had been submitted to 500 μM CaCl2 solution, in the absence or the presence of {20} μM Al3+, pH 4.2, in hydroponic system for 1, 3, and 5 days, total RNA was extracted from shoots using TRIzol® reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. RNA from roots was extracted using a LiCl method (). The samples were treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Total RNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Uniscience), and RNA integrity was verified in agarose gel electrophoresis. The synthesis of first strand cDNA was accomplished using the extracted RNA as template and the RevertAidTM First Strand cDNA Synthesis kit (Thermo Fisher Scientific). These steps were all performed according to the manufacturer’s instructions. The qPCR was carried out using Platinum® SYBR® Green PCR SuperMix-UDG with ROX (Invitrogen, Carlsberg, CA, USA) with synthesized single-stranded cDNA as template, using the protocol recommended by the StepOnePlusReal-Time PCR Systems (Applied Biosystems). The primers used in the qPCR are described in Supplementary Table .Relative gene expression levels were calculated using the q-Gene (). SiEF1-α; SiSUI, SiCAC, and SiCUL were used as reference genes () and the geometrical mean of the relative quantities (RQs) was calculated using BestKeeper software (). Individual amplification efficiencies were established with LinRegPCR v.2013.0 using a window-of-linearity (). The experiment was performed using three biological replicates, with 40 plants each. […]

Pipeline specifications

Software tools BestKeeper, LinRegPCR
Application qPCR
Organisms Setaria viridis, Sorghum bicolor, Ilex paraguariensis, Zea mays, Triticum aestivum
Chemicals Aluminum, Hematoxylin