The analyses of plastid gene transcription Hordeum vulgare in darkness

Experiments were performed with the leaves of barley plants (Hordeum vulgare L.). The seeds were germinated in soil in the climate-controlled chamber in darkness at 20-22°C.First true leaves detached from 6-day-old seedlings were used. The age was counted from the moment of seedling emergence above the soil surface. Plastid isolation and run-on transcription in plastid lysates were performed as described Zubo and Kusnetsov, 2008. Etioplasts were isolated from etiolated plants under dark green light and purified in the discontinuous gradient of Percoll (40 and 70%). In vitro transcription was run for 10 min at 25°C in the lysates of 5 . 107 chloroplastsin buffer. Fragments of the tested genes (1 μg) were loaded on the nylon membrane Hybond-N+ (Amersham Pharmacia Biothech, England) in two replications. After their hybridization with 32P-labeled RNAs produced in the reaction of run-on transcription in vitro, radioactive signals were scanned using Phosphorimager Typhoon Trio+ (GE Healthcare, USA).The rate transcription of 16 chloroplast gene was analyzed. First of all, genes, product which plays supreme role in photosynthesis realization - photosystem I - psa (psaA and psaB), photosystem II - psb (psbA, psbD and psbK), gene of large subunit of Rubisco (rbcL), ATP synthase complex - atp (atpB) and F subunit NADPH of plastochinon oxidoreductase - ndhF. Among «housekeeping» genes the transcription of gene, which code β subunit of plastid bacterial-type RNA polymerase (rpoB), 16S and 23S rRNA genes (rrn16 and rrn23), also tRNA genes - Gly and Tir (trnE-Y) was investigated. The results obtained showed that the rate of chloroplast gene transcription in the first barley leaves was significantly dispersed. The highest transcription was marked to genes, which coded protein of photosystem II (psbA, psbB and psbD), large subunit of Rubisco (rbcL), ATP-synthase (atpB), ribosomal proteins (rrn16, rrn23 and rps16), also tRNA genes (trnE/trnY).

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