Computational protocol: Transcriptional and physiological changes in the S assimilation pathway due to single or combined S and Fe deprivation in durum wheat (Triticum durum L.) seedlings

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Protocol publication

[…] The available Arabidopsis sequences of genes coding for high-affinity transporters and enzymes involved in sulphate assimilation and reduction (Supplementary Table S1 available at JXB online) were used as queries in a BLAST search of public databases of wheat expressed sequence tags (ESTs): DFCI wheat gene index database (TaGI, version 12) and NCBI. The identified non-redundant sequences were used as templates for 5’ and 3’ RACE (rapid amplification of cDNA ends) extensions using the 5’/3’ RACE kit from Roche following the manufacturer’s instructions. Total RNA was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions, and its concentration and quality were checked as reported in and described in detail in the Supplementary Appendix S1 at JXB online. RACE products were amplified using 2 μg of a pool of total RNA from roots and shoots of the durum wheat cv Svevo. The 5’ and 3’ RACE products were cloned and validated by sequence analysis; the corresponding full-length cDNAs of each identified wheat gene involved in S metabolism were cloned by reverse transcription–PCR (RT–PCR) using total RNA from roots and shoots of cv. Svevo and specific primers designed on the basis of the 5’- and 3’-untranslated regions (UTRs) (Supplementary Table S2). PCRs were performed using 2 μl of the reverse transcription reaction with the GC-Rich PCR System from Roche following the manufacturer’s instructions.RACE and full-length cDNA products were cloned into the pGEMT easy plasmid vector (Promega). Sequencing was performed on both strands by the ABI PRISM 377 capillary sequencer (PE Applied Biosystem) using an ABI Prism Dye Terminator sequencing kit (PE Applied Biosystem) and either vector- or sequence-specific primers. Full-length cDNA sequences were deposited in the DDBJ/EMBL/GenBank nucleotide sequence databases; accession numbers are indicated in . A two-letter code (Td=Triticum durum) followed by the suffix of the corresponding transporters or enzyme involved in S metabolism was assigned to each sequence. The predicted protein sequences were analysed by searching for conserved motifs in CDD, Pfam HMMs, InterPro, and SMART databases; their subcellular locations were predicted by Target P1.1 and ChloroP 1.1. […]

Pipeline specifications

Software tools InterPro, ChloroP
Databases DDBJ Pfam Full Length cDNA
Application Protein sequence analysis
Organisms Triticum aestivum
Diseases Deficiency Diseases
Chemicals Adenosine Triphosphate, Iron