Computational protocol: Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes

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Protocol publication

[…] To clone homologues of PvPAP3, AtPAP10, and AtPAP26 in stylo, primers (SgPAP7/10/26-EST-F and SgPAP7/10/26-EST-R, Supplementary Table S1) were designed separately according to the conserved motif sequences of PvPAP3 (NCBI accession FJ464333.1), AtPAP10 (NCBI accession NM_127196.3), AtPAP26 (NCBI accession NM_122874.3), and three GmPAPs (NCBI accessions NM_001254279.1, NM_001253997.1, NM_001249748.1). Three fragments were successfully amplified by PCR from the cDNA of TPRC2001-1 plants subjected to P deficiency, and then cloned into the pMD18-T vector (Takara, Japan). Subsequently, the 5′ and 3′ terminals of the three PAP fragments were amplified from a TPRC2001-1 RACE cDNA library constructed with the SMARTer™ RACE cDNA amplification kit (Clontech, USA) using specific primers paired with the universal primers RACE-UPM and RACE-NUP (Supplementary Table S1). After separately cloning 5′-terminal and 3′-terminal cDNA products into the pMD18-T vector, full-length sequences of SgPAP7, SgPAP10, and SgPAP26 were generated through the MEGA 5 program. The nucleotide sequences of these three SgPAPs were deposited in NCBI and assigned the accession numbers KU315544, KU315545, and KU315546 for SgPAP7, SgPAP10, and SgPAP26, respectively. Multiple sequence alignments and phylogenetic trees were constructed in ClusterX and MEGA 5, respectively. Signal peptides were predicted by SignalP 4.1 (http://www.cbs.dtu.dk/services). The transmembrane topology of the proteins was predicted by MEMSATSVM (http://bioinf.cs.ucl.ac.uk/web_servers/). […]

Pipeline specifications

Software tools MEGA, SignalP
Application Phylogenetics
Chemicals Phlorhizin, Phosphates, Phosphorus