Computational protocol: Arbuscular mycorrhizal symbiosis alters the expression patterns of three key iron homeostasis genes, ZmNAS1, ZmNAS3, and ZmYS1, in S deprived maize plants

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Protocol publication

[…] Performing Blast searches we identified the cDNA sequences of all recorded ZmNAS and ZmYS genes. Primers were designed for a representative of each class of the ZmNAS gene family as well as for ZmYS1 (MaizeGDB: GRMZM2G156599). ZmNAS1;1 (MaizeGDB: GRMZM2G385200) and ZmNAS3 (MaizeGDB: GRMZM2G478568) were chosen for class I and II respectively. Real-time PCR primers were designed to amplify 100–200 bp fragments in the 3′untranslated regions, using the Primer Blast tool of NCBI. All primers were designed for 60°C annealing temperature and their sequences are as follows: ZmNAS1;1: Forward 5′-GGAACTTTTGAGCACCTATGCG-3′ and Reverse 5′-CACTTCACAATGCATAGCATCGAAT-3′; ZmNAS3: Forward 5′-CGTGTCTACACCACATGCGT-3′ and Reverse 5′-TCGGACTTCGACTTCTACCCT-3′; ZmYS1: Forward 5′-GTCTTCCATTCTCGCTCTGG-3′ and Reverse 5′-CAACCAACCACAGTTGATGC-3′. The gene of ubiquitin was used as an internal control and the target was detected by the following primers-pair: ZmUBQ (NCBI: NM_001138130): Forward 5′-TGTCTTCATGGCCAACCACT-3′ and Reverse 5′-GCTTGATAGGTAGGCGGGTG-3′. [...] Total nucleic acids were extracted from root and shoot samples using the Phenol-Chloroform protocol (Brusslan and Tobin, ) and were treated thereafter with Recombinant DNase I (RNase-free, Takara Bio Inc) in order to get the total RNA of each sample. An amount of 500 ng of RNA was reverse-transcribed using the PrimeScript RT reagent (Perfect Real Time, Takara Bio Inc).Measurements of real-time RT-PCR were performed using KAPA SYBR FAST Master Mix (KAPA Biosystems) in the MxPro Mx3005P thermocycler (Stratagene, USA). The real-time RT-PCR was performed according to the respective protocol of the kit, using optical 96-well plates with the following PCR program: 95°C for 10 min, 40 cycles of 95°C for 30 s and 60°C for 1 min and the final cycle of 95°C for 1 min and 60°C for 30 s and 95°C for 30 s. Melting curve analysis was carried out after each amplification to exclude unspecific amplifications from the analysis. PCR amplification efficiencies were obtained using the LinRegPCR software (Ruijter et al., ). The relative expression ratios were calculated with the mathematical formula of Pfaffl (), using as reference the gene of ubiquitin and as targets the genes of ZmNAS1, ZmNAS3, and ZmYS1. As control the samples of day 30 (for the samplings before sulfur supply) or day 60 (for the samplings after sulfur supply) of the respective treatment were used. […]

Pipeline specifications

Software tools Primer-BLAST, LinRegPCR
Databases MaizeGDB
Application qPCR
Organisms Zea mays, Rhizophagus irregularis
Diseases Protein S Deficiency
Chemicals Iron, Methionine