Construction of a cDNA library

About 30 seeds of Catuai red were allowed to germinate on wet blotting paper. The roots (about 15 mm for a total of 3.5 g) were cut and immediately frozen in liquid nitrogen. Following the grinding of the tissue, the total RNA was extracted using the protocol described by Chomczynski e Sacchi (1986) and the polyadenylated mRNA was purified by oligo-dT column chromatography by standard techniques. The first strand of cDNA was synthesised by reverse transcriptase and an anchored oligo-dT-NotI primer. After completion of the second strand, using the Gubler and Hoffman strategy, the cDNA fragments were repaired with T4 DNA Polymerase, ligated to BstXI non-palindromic adapters, digested with NotI and size-fractionated on CLB4 spun column. The cDNA was directionally cloned into a BstXI, NotI digested pcDNAII plasmid vector and the ligation reaction product was used for transforming electro-competent E. coli DH10B cells.

EST preparation

Following the construction of the cDNA library, the single clones were isolated and collected. Each colony was transferred to 384 well plates and allowed to grow on SOB culture medium supplemented with ampicillin (60 µl per well), at the temperature of 37°C. Ten plates were prepared for a total of 3840 bacterial clones. About 200 µl of bacterial culture were transferred with a 384 tooth comb to a 384 well PCR plate together with 20 µl of reaction mix. The cells were lysed during the first denaturation phase at 95°C. The DNA released by the cells was amplified under the following conditions: 95°C for 5’, first cycle; 95°C x 30”, 55°C x 30”, 72°C x 2’30” for the following 35 cycles. At the end of the reaction, 5 µl of mix underwent electrophoresis on 1% agarose gel to check the quality of the amplification and the length of the amplicons. The DNA sequencing was performed only on the amplicons longer than 500 bp.

Bioinformatic analysis of sequences and database construction

Sequences have been corrected and assembled using the Seqman programme of the informatic package DNAStar (Lasergene). The following parameters have been applied: End Trimming High, Assembling Match Size 50 with a Minimum Match Percentage 90. These parameters are very high: we preferred to have a higher number of contigs rather than create informatic chimeras. The search for similarities was facilitated by a series of Perl scripts created by our team. We have used the netblast programme, in that it allowed us to use the NCBI database bypassing the graphic interface on the web. To identify the homologies, all the contigs were compared to nucleotide sequences, including the ESTs stored in specialised databases, and to polypeptide sequences.