Computational protocol: Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook

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Protocol publication

[…] To isolate the potential SERK and WOX genes, the transcriptome database of C. lanceolata produced by , , and was screened using the NCBI tblastn algorithm using the SERK homologs in Arabidopsis, Medicago, and rice (see Supplementary Table ), and the WOX homologs in P. abies (; ) and Arabidopsis () (see Supplementary Table ). The potential genes were named on the basis of their alignment to Arabidopsis (see Supplementary Table ). The unrooted neighbor-joining phylogenetic trees were generated from the deduced protein sequences using MEGA5.2 with the help of the Jones-Taylor-Thornton (JTT) model in combination with the gamma-distributed rate model (JTT+G; gamma = 0.97). Bootstrap values from 1,000 replicates were indicated at each node.Tissues at different developmental stages were collected. Total RNA was extracted and purified following the methods previously described by . RNA (1 μg) was reverse-transcribed with oligo (dT) and random hexamer primers using a reverse transcription system (Promega). Verification was performed using qRT-PCR.qRT-PCR was carried out using the LightCycler 480 System (Roche Applied Science). Each reaction was performed in a 20-μL final volume containing 10 μL of 2× LightCycler 480 SYBR Green I Master, 0.5 μL of each homolog-specific primer pair (see Supplementary Table ) at 100 nM, 5.0 μL of diluted cDNA template, and 4.0 μL of ddH2O. Each reaction was run in triplicate with the appropriate negative controls. Amplification was conducted under the following conditions: activation for 5 min at 95°C, followed by 40 cycles of 10 s at 95°C, 15 s at 60°C, and 15 s at 72°C. Fluorescence detection was performed after the extension step. The melting curve, with a temperature gradient from 60 to 95°C, was used to further investigate the specificity of each qRT-PCR reaction, and the presence of a single PCR product was also verified by 2% agarose gel electrophoresis. The eIF-3 housekeeping gene was selected as the endogenous reference gene for the relative PCR quantification (). […]

Pipeline specifications

Software tools TBLASTN, MEGA
Applications Phylogenetics, Amino acid sequence alignment
Organisms Cunninghamia lanceolata, Caenorhabditis elegans, Arabidopsis thaliana
Chemicals Abscisic Acid, Polyethylene Glycols