سجل مستودعي · ncbi-prjna407796

Arabidopsis thaliana Transcriptome or Gene expression

Biological material involved arabidopsis WT (Col), the knockout T-DNA insertion (SALK_150614) mutant (namely KOERF109) and over-expression lines of ERF109 gene (CS2102255) (OEERF109) of locus AT4G34410 provided by the SALK Institute, Genomic Analysis Laboratory (SIGnAL) (http://signal.salk.edu/tdnaprimers.2.html). Plantlets were grown from seed in a growth chamber for two weeks under the following growth conditions. First, seeds of the three genotypes were surface sterilized, sown in Petri dishes containing MS medium. The plates were kept in the dark at 4°C for 2 days and then shifted to 21 ± 2°C (day/night) under light intensity of ~175 umoles m-2sec-1 and a 16-h-light/8-h-dark cycle where plantlets were allowed to grow for 12 more days. Knockout mutant was screened for homozygosity by standard PCR approach, while seeds of the over-expression line were homozygous. Sequences of PCR primers and reaction conditions were recovered from Arabidopsis database (TAIR, http://www.arabidopsis.org/).Salt stress experiments were conducted at the laboratories of the Department of Biological Sciences, KAU, Jeddah, Saudi Arabia. In order to harvest transcriptomes under salt stress, 2-wk-old plantlets of KOERF109 and OEERF109 lines as well as the WT (Col) with homogeneous performance were transferred to pots (9 cm) filled with soil mix (1 soil : 1 vermiculite), where salt stress experiment started. Two-wk-old control untreated and salt-stressed plantlets were allowed to grow at the above mentioned growth conditions. Control plantlets were irrigated daily with deionized double distilled water and allowed to grow for two more weeks. While, salt-stressed plantlets were initially irrigated daily with salt concentration of 50 mM NaCl for one week. Then, two incremental increases of salt stress was made as recommended by Munns41. An increase of 75 mM NaCl was made for 3-wk-old plantlets, which were left to grow for one more week. Another increase of 75 mM NaCl was made for 4-wk-old plantlets which is the target salt concentration (200 mM NaCl) for RNA-Seq analysis. At the same day of reaching the target salt concentration, leaf samples of the 4-wk-old plantlets were harvested 2 and 12 h. Leaf samples of the control unstressed 4-wk-old plantlets were harvested concurrently with those harvested 12 h after salt treatment.