Computational protocol: Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis

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Protocol publication

[…] Members of the IPT family in the genomes of 12 selected sequenced green plants and algae (Arabidopsis thaliana, Chlamydomonas reinhardtii, Chlorella variabilis, Cyanidioschyzon merolae, Micromonas pusilla, Micromonas sp., Oryza sativa, Ostreococcus lucimarinus, Ostreococcus tauri, Selaginella moellendorffii, Volvox carteri, and Zea mays) were identified using protein clustering as described previously (; ). The resulting candidate list was collated with the clusters in the Phytozome plant gene family database () and previous descriptions of the IPT family in Arabidopsis, rice, and maize. To provide further taxonomic coverage, additional members of the IPT family were added based on their InterPro protein domain annotation (). Using the InterPro database web interface, proteins containing the IPT protein domain (PF01745; IPR002627) from selected cyanobacteria, Alveolates, Euglenozoa, Stramenopiles, and Agrobacterium tumefaciens were included into the candidate set. Sequence data are available as a supplementary file at JXB online. To derive a high-quality alignment, the manually curated alignment of the TRIT1 tRNA isopentenyltransferase family from the TreeFam database (TF315069) was used as a starting point for profile alignment using T-Coffee 8.14 (). Based on the resulting full alignment (5517 amino acid columns), a Neighbor–Joining (NJ) guide tree was calculated using the Scoredist matrix () implemented in a modified version of QuickTree () applying bootstrap resampling with 1000 replicates. The resulting tree was used to reorder the alignment and manually curate and clip it using Jalview () by removing phylogenetically uninformative sites and reducing the full alignment to a clipped and reduced alignment spanning 203 residues and comprising 97 proteins. A full list of all IPT family members in this curated alignment is provided in Supplementary Table S1 at JXB online. The resulting clipped protein alignment was used to infer the IPT gene phylogenies using QuickTree and MrBayes (). MrBayes was run with parameter settings lset nst=6 rates=invgamma Ngammacat=4; prset aamodelpr=mixed; mcmc stopval=0.01 stoprule=yes nruns=2 nchains=128 ngen=5000000 printfreq=1000 samplefreq=100 temp=0.2 starttree=random relburnin=yes burninfrac=0.25 savebrlens=yes. […]

Pipeline specifications

Software tools InterPro, T-Coffee, QuickTree, Jalview, MrBayes
Databases Phytozome
Applications Phylogenetics, Protein sequence analysis
Organisms Physcomitrella patens