Computational protocol: Molecular Cloning, Recombinant Expression and Antifungal Activity of BnCPI, a Cystatin in Ramie (Boehmeria nivea L.)

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Protocol publication

[…] The online ORF finder program of NCBI (https://www.ncbi.nlm.nih.gov/orffinder/) was used to search the entire ORF sequences of BnCPI, while the online BLAST program of NCBI was used to search protein homology. Theoretical molecular weight (MW) and isoelectric point (pI) for BnCPI were predicted using the ExPASy tool (http://web.expasy.org/compute_pi/); signal peptide prediction was performed using SignalP 4.1 (http://www.cbs.dtu.dk/services/SignalP/); and secondary structure was predicted using SOPMA []. The conserved domain was searched against the NCBI Conserved Domain Database [].A set of primers (cpif: 5′-CGC AGA AAA GTA AAA GCA-3′ and cpir: 5′-TCC ACC AAA GAC GAA TGA-3′) was assigned for PCR amplification of ORF fragments in different ramie cultivars. Primers cpif and cpir were located upstream of the start codon and downstream of the stop codon of the ORF, respectively. PCR was performed in an ETC-811 PCR instrument (Eastwin, Beijing, China). The reaction mixture contained 25 μL of 2× Taq PCasterMix (Aidlab, Beijing, China), 4 μL of each primer (10 μmol/μL), 2 μL of genomic DNA or cDNA (10 μg/μL), and the total volume was adjusted to 50 μL with ddH2O. The PCR amplification procedure consisted of 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 1 min, and extension at 72 °C for 1 min, with final extension at 72 °C for 10 min. The PCR products were separated by electrophoresis on 1.5% agarose gels and visualized by staining with Gold View (Applygen, Beijing, China). Amplicons were sequenced by Tsingke Company (Tsingke, Beijing, China). BnCPI and phytocystatins from other plants downloaded from the NCBI website were aligned using MEGA 6.0 software []. The resulting alignment was trimmed and a phylogenetic tree was constructed using the neighbor-joining method with 100 bootstrapping. The deduced amino acid sequence of BnCPI was also aligned with those of other phytocystatins available in the EMBL-EBI database ClustalW2 (http://www.ebi.ac.uk/Tools/msa/muscle/), set at default parameters. […]

Pipeline specifications

Software tools Open Reading Frame Finder, SignalP, SOPMA, MEGA, Clustal W
Databases CDD ExPASy
Applications Phylogenetics, Protein structure analysis
Organisms Hevea brasiliensis, Ricinus communis, Escherichia coli BL21(DE3)
Chemicals Amino Acids, Glycine, Salivary Cystatins