Random sequencing of cDNAs and identification of mRNAs
2001
Anderson, James V. | Horvath, David P.
As a first step toward developing a genomics-based research program to study growth and development of underground adventitious shoot buds of leafy spurge, we initiated a leafy spurge expressed sequence tag (EST) database. From the approximately 2,000 clones randomly isolated from a cDNA library made from a population containing growth-induced underground adventitious shoot buds, we have obtained ESTs for 1,105 cDNAs. Approximately 29%% of the leafy spurge EST database consists of expressed genes of unknown identity (hypothetical proteins), and 10%% represents ribosomal proteins. The remaining 60%% of the database is composed of expressed genes that show BLASTX sequence identity scores of ≥≥ 80 with known GenBank accessions. Clones showing sequence identity to a Histone H3, a gibberellic acid-responsive gene, Tubulin, and a light-harvesting chlorophyll a/b-binding protein were shown to be differentially expressed in underground adventitious shoot buds of leafy spurge after breaking of dormancy. RNA encoding a putative cyclin-dependent protein kinase (CDK)-activating kinase, a gene associated with cell division, and Scarecrow-like 7, a gene involved in GA signaling, were present at similar levels in dormant and growth-induced underground adventitious shoot buds. These data show how even a small EST database can be used to develop a genomics-based research program that will help us identify genes responsive to or involved in the mechanisms controlling underground adventitious shoot bud growth and development.Nomenclature: Amp, ampicillin; CAK, CDK-activating protein kinase; CDK, cyclin-dependent protein kinase; EST, expressed sequence tag; LB, Luria––Bertani broth; Lhcb, light-harvesting chlorophyll a/b-binding protein; NCBI, National Center for Biotechnology Information; SSC, sodium chloride sodium citrate solution.
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