Construction and screening of a pea root cDNA library

Abstract

A cDNA library was synthesized using poly(A)(^+) RNA purified from the total RNA from roots of pea (Pisum sativum L.). Ten representative clones encoding abundant root proteins were isolated from the library after screening using colony hybridization method which were probed by the root cDNAs. Freeze elation technique was used to extract three different partial pea root-specific genes which have been cloned in plasmid vectors pUC18 previously. These purified DNAs were radiolabelled and then used to probe with the library. One of the probes namely pPR179 was found to be highly specific and hybridized strongly to some of the root cDNA clones. This allowed full-length cDNAs that encoded root-specific pro- tein(s) to be identified for subsequent analysis. Restriction patterns of the pea root cDNA-plasmid pUC19 recombinants revealed some artefactual cDNAs were synthesized and possible explanations were attempted.