Isolation of Callus-specific mRNAs from Differentiating Embryogenic Somatic Calli of Pimpinella brachycarpa by cDNA-AFLP

摘要

We used cDNA-AFLP to analyze gene expression profiles during somatic embryogenesis and to isolate stage-specific genes in Pimpinella brachycarpa. Ninety- six combinations of AFLP primers produced 3,785 fragments, of which 478, 403, and 436 were specific to calli, somatic embryos, and plants, respectively. 262 fragments were shared by callus and somatic embryo, 324 by somatic embryo and plant, and 222 by callus and plant. The number of fragments common to all three stages was 1,660. Seventy-five of the 478 callus-specific fragments were cloned and sequenced and their functions sought in the GenBank database. Thirty-three of them showed significant homology to GenBank data base entries and twelve of these were homologous to genes with products of known function such as ATPases, tubulins, kinases, transcription factors, ubiquitins, and ankyrin. Callus-specific expression was confirmed by performing RT-PCR with six of the twelve fragments. Since the somatic embryo is induced by auxin (Thidiazuron), possible down regulation of these genes by auxin is also discussed.