Isolation of cDNA encoding cytokinin-binding proteins in maize.

Two approaches were used to enrich RNA isolated from etiolated maize (Zea mays L.) seedlings with mRNAs for the receptor cytokinin-binding protein CBP70. The first method was immunoaffinity chromatography of polysomes using immobilized monoclonal antibodies against CBP70, and the second one was polysome affinity chromatography on immobilized zeatin riboside. RNA obtained by both methods and enriched with sequences encoding the cytokinin receptor was used for constructing cDNA libraries in the lambda MOSElox expression vector in the Escherichia coli cells, strain B834(DE3). Clones were screened for CBP70 synthesis using monoclonal antibodies against this protein. Among 15 000 clones from the cDNA library constructed by the first method and 5000 clones from the cDNA library obtained by the second method, 38 and 12 clones, respectively, cross-reacted with antibodies against CBP70. Immunoblotting of polypeptides From lysates of these positive clones detected the polypeptides close to CBP70 in their mol wts. Thus, cDNAs corresponding to mRNAs for the cytokinin-binding protein were obtained by two independent methods. The developed experimental approaches can be recommended for enriching the total RNA fraction with infrequent mRNAs.