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United States Department of Agriculture

Agricultural Research Service

Title: Random Sequencing of Cdnas and Identification of Mrnas

Authors
item Anderson, James
item Horvath, David

Submitted to: Weed Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 18, 2001
Publication Date: August 1, 2001
Citation: Anderson, J.V., Horvath, D.P. 2001. Random sequencing of cDNAs and identification of mRNAs. Weed Science. 49:590-597.

Interpretive Summary: We have initiated a genomics-based research program within our research unit. To accomplish this task, it was necessary to have a source of genetic material to work with. Since there is little genetic material available for perennial weeds, we have initiated the development of an expressed sequence tag (EST)-database for leafy spurge. To initiate an EST-database, we have isolated 2000 cDNA clones from a leafy spurge cDNA library to growth-induced, underground adventitious shoot buds (UABs). DNA sequence to 1105 purified plasmids from the isolated cDNAs have been submitted to GenBank for a BLASTX search. The BLASTX search will find the best identity match to already known genes within the database at the National Center for Biological Information. Sequence identity matches to our EST-database revealed a number of genes that are likely to be differentially expressed in growth-induced leafy spurge UABs. We have used some of the clones from our EST-database to test for differential patterns of expression in growth-induced UABs, foliar meristematic tissue, and in mature leaf tissue. The data reported in this manuscript do indicate that clones within our EST-database show differential patterns of expression and may be regulated by different signal-transduction pathways. This data also adds support for our hypothesis that lack of growth in UABs may be due to a blockage of cell division resulting from interactions between signaling pathways controlling dormancy and those controlling the cell cycle.

Technical Abstract: As a first step towards developing a genomics-based research program to study growth and development of underground adventitious shoot buds (UABs) of leafy spurge (Euphorbia esula L.), we have initiated a leafy spurge EST-database. From the approximately 2000 clones randomly isolated from a cDNA library made from growth-induced UABs, we have obtained expressed sequence tags (EST) to the first 1105 isolated cDNA. Approximately 29% of the leafy spurge EST-database is composed of expressed genes of unknown identity (hypothetical proteins) and 10% ribosomal proteins. The remaining 60% of the leafy spurge EST-database is composed of expressed genes which show BLASTX sequence identity scores of ò80 for known GenBank accessions. Clones showing sequence identity to a histone H3, a CDK-activating kinase (CAK), a gibberellic acid-responsive gene (GASA), tubulin, and a light-harvesting chlorophyll a/b-binding protein (Lhcb1) were shown to be differentially expressed in leafy spurge tissues of differing morphology and development. CAK, a gene associated with cell division, is constitutively expressed in dormant and growth-induced UABs. Histone H3, another cell division gene, is not expressed in dormant UABs, but does show expression within 24-36 h after removal of foliar tissue, and also is expressed in foliar meristematic tissue. GASA expression parallels that of histone H3. The expression of Lhcb1 is lower in dormant UABs but shows increased expression in UABs of defoliated plants. These data shows how our leafy spurge EST-database can be used to develop a genomics-based research program that will help us to identify mechanisms controlling UAB development which could lead to novel control strategies.

Last Modified: 9/10/2014
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