S.lycopersicum R11-12 leaves inoculated with Pst(avrPto)
5174 sequences from 5127 clones
Average Sequence Length:
465.8 (Standard deviation 101.4)
For each library four-week old plants were inoculated with the Pst(avrPto)strain and the leaves were harvested. Two plants were used for each time-point as follows: High inoculation level: 10^8 cfumL of Pst(avrPto). Equal amounts of leaves were harvested at 0, 2, 4, 6, 8 hrs after inoculation. Low inoculation level: 10^5 cfumL of Pst(avrPto). Equal amounts of leaves were harvested at 0, 12, 24, 36, 48 hrs after inoculation. High bacterial titer gives a hypersensitive response on R11-12 at 10-12 hours after inoculation. Low bacterial titer gives no HR on R11-12, but results in disease symptoms beginning at about 48 hrs after inoculation. Keeping leaves from each line separate, equal amounts of leaves from each time-point were pooled and used to extract polyA RNA using Promegas polyATract mRNA isolation system. Stratagenes Zap-cDNA synthesis kit was used to prepare cDNA. The cDNA was cloned into Uni-ZAP XR vector using the EcoRI site at the 5-end and the XhoI site at the 3-end. Over 10^6 pfus were obtained for each library. When a sample of these were plated on NZY agar plates containing IPTG and X-gal, the white:blue colony ratio was about 100:1. Mass excision was performed on 10^7 pfus from each library. After mass excision, twenty-five clones from each library were picked randomly and characterized. All 50 clones contained inserts with sizes ranging from 0.5 to more than 3 Kb (avg=1Kb). We sequenced the 5-ends of 14 random clones (7 from each library).
He,X., DAscenzo,M., Lyman,J. and Martin,G.
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