Computational protocol: Dual mechanisms regulating glutamate decarboxylases and accumulation of gamma-aminobutyric acid in tea (Camellia sinensis) leaves exposed to multiple stresses

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Protocol publication

[…] Three GAD enzymes (CsGAD1, 2 and 3) were selected from a tea transcriptome database. The C-proximal regions of CsGADs were truncated (detailed information is shown in the Results section). Total RNA was isolated from finely powdered plant tissues (100 mg) using a Spectrum Plant Total RNA Kit (Sigma-Aldrich, St. Louis, CA, USA). The first strand cDNA was synthesized using the Reverse Transcription System (Promega, Madison, WI, USA). The full length open reading frames (ORF) were amplified from first strand cDNA with primers listed in . The resulting PCR products were purified and subcloned into the pGEM-T vector (Promega, Madison, WI, USA) and sequenced. Sequences were aligned using the ClustalX program. The phylogenetic tree was calculated by neighbor-joining algorithms and generated by MEGA 5 software. Analysis of the CaM-binding domain sequences utilized the high-performance computational capabilities of the Helix Systems at the National Institutes of Health, Bethesda, MD ( […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Camellia sinensis, Homo sapiens, Arabidopsis thaliana
Chemicals gamma-Aminobutyric Acid, Glutamic Acid