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The bottom of each box was covered with foil to induce vigorous and vital roots in darkness.
After 6 weeks, when plantlets were growing under balanced physiological conditions, they were subjected to various stress treatments.
The PCR-cycling conditions (Biometra) consisted of an initial denaturation step of 95 °C for 15 min followed by 35 cycles of 60 s at 95 °C, 60 s at annealing temperature (60 °C), and 2 min at 72 °C. For Southern blot analyses, 3 μg of genomic DNA was digested with 40 units of the restriction endonucleases RV (Roche) according to the procedure elaborated and described previously (Maghuly et al. Three-centimeter-high shoots were transferred to Phytatray II (P5929-50EA SIGMA, St.
Louis, MO) boxes containing 100 ml rooting medium (Druart 1990).
The first-strand c DNA synthesis was performed on 2 μg total RNA treated with DNase I (Roche) using Super Script according to the supplier’s recommendation.
The reverse transcription (RT) reaction was diluted 1/10 to reduce inhibitory effects of the RT buffer system.
The aim of this study is to find out whether expression of these proteins is regulated in parallel or independently and to compare the expression profiles of are encoded by at least two to four genes, which might be indicative for the expected variability and presence of isoforms.
They were removed, immediately frozen in liquid nitrogen and stored at −70 °C until the isolation of total RNA.
Real Time q PCR data showed that both genes respond differently to various stresses.
Furthermore, a high variation in transcription levels of both genes was observed in leaf tissues, while in roots both genes were expressed at a lower extent and down-regulated. In higher plants, unlike in animals, Ca Ms are present as multiple isoforms even within species.
Calcium-binding proteins and pathogenesis-related (PR) proteins are crucial components of the inducible repertoire of plant stress and defence.
Considering the important role played by calmodulin (Ca M) and lipid transfer protein (LTP) in mediating plant signal transduction, the present study investigated the expression of (PIS) under various abiotic stress conditions.