Computational protocol: Overexpression of the alfalfa WRKY11 gene enhances salt tolerance in soybean

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Protocol publication

[…] MsWRKY11 was cloned according to the sequence information of the transcriptome of the alfalfa cultivar Zhaodong under salt and temperature stress. Total RNA was isolated from alfalfa seedlings treated with NaCl solution using an RNA extraction kit (TianGen Biotech, Beijing, China) according to the manufacturer’s protocol. The RNA was reverse-transcribed into cDNA using a PrimeScript RT reagent kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Reaction volumes (20 μL) contained 1 μL of Oligo20, 10 μL of RNA, 1 μL of RNase-free double-distilled water, 4 μL of 5× RT buffer, 2 μL of dNTP mix, 1 μL of RNase inhibitor, and 1 μL of reverse transcriptase. The amplification was run at 30°C for 30 min, 42°C for 20 s, 99°C for 5 min, and 4°C for 5 min (GeneAmp PCR System 9700, USA).The cloning primers were designed according to the sequence information of the alfalfa transcriptome under salt stress by using Primer 5.0 software (primer 1: 5′-CTACCGGATCTACAACCATTCTAGAGC-3′, primer 2: 5′-CGAGCTCTCAAAGAGGCTGAGATAT-3′). The cDNA was diluted tenfold and used as the PCR template, and the MsWRKY11 gene was amplified in 50 μL reactions containing 5 μL of cDNA, 5 μL of 10× dNTP mix, 4 μL of primer mix (primers 1 and 2), 0.2 μL of Ex Taq DNA polymerase (5 U/μL), and 31.8 μL of RNase-free double-distilled water. The cycling protocol consisted of the initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and elongation at 72°C for 1 min; final elongation at 72°C for 10 min; and extension at 16°C for 2 h (GeneAmp PCR System 9700). The DNA fragments were recovered from the agarose gel using a DNA Recovery Kit (TianGen Biotech).The PCR products were sent to Sangon Biotech Co., Ltd. (Shanghai, China) for sequencing. The amino acid sequence was inferred by DNAMAN software (Lynnon LLC, San Ramon, CA, USA). Sequences from other species were obtained from GenBank (https://www.ncbi.nlm.nih.gov/protein) and aligned with the sequence for MsWRKY11 in ClustalX [] (). The neighbor-joining method was used to generate the phylogenetic tree. The protein sequences of MsWRKY11, GmWRKY65, GmWRKY11, and AtWRKY11 were analyzed using DNAMAN. […]

Pipeline specifications

Software tools DNAMAN, Clustal W
Application Phylogenetics
Organisms Glycine max
Chemicals Abscisic Acid, Chlorophyll, Hydrogen Peroxide, Malondialdehyde, Superoxides, Zinc