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Title: SUBTRACTIVE CDNA LIBRARIES IDENTIFY DIFFERENTIALLY EXPRESSED GENES IN DORMANT AND GROWING BUDS OF LEAFY SPURGE (EUPHORBIA ESULA)

Author
item JIA, YING - NDSU
item Anderson, James
item Horvath, David
item Gu, Yong
item LYM, RODNEY - NDSU
item Chao, Wun

Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/23/2006
Publication Date: 7/1/2006
Citation: Jia, Y., Anderson, J.V., Horvath, D.P., Gu, Y.-Q., Lym, R.G., Chao, W.S. 2006. Subtractive cDNA libraries identify differentially expressed genes in dormant and growing buds of leafy spurge (Euphorbia esula). Plant Molecular Biology. 61:329-344.

Interpretive Summary: A genomics approach was applied to identify and clone genes associated with dormancy and growth in the root and crown buds of leafy spurge. Two subtracted (a forward and a reverse) cDNA libraries were developed for this work. The forward library contains genes preferentially expressed in growing buds and the reverse library contains genes preferentially expressed in dormant buds. After library screening, 521 non-overlapping genes were obtained. The expression of these genes was examined based on macroarray and semi-quantitative RT-PCR analysis. Many differentially-regulated genes during bud development were identified. Some of these genes have unknown or hypothetical functions while others are known to play important roles in molecular functions such as cytochrome P450 and lipid transfer protein.

Technical Abstract: Two subtractive cDNA libraries were developed to study genes associated with bud dormancy (reverse library) and initiation of shoot growth (forward library) in leafy spurge. To identify unique sequences represented in each library, 15744 clones were screened to reduce the level of redundancy within both libraries. A total of 516 unique sequences were obtained from 2304 minimally redundant clones. Radio-active probes developed from RNAs extracted from crown buds of either intact (para-dormant control) or a series of growth-induced (2 h, 2, and 4 d after decapitation) plants were used to identify differentially expressed genes by macroarray analysis. Semi-quantitative RT-PCR was used to confirm results obtained by macroarray analysis and to determine the expression profiles for other transcripts identified within the subtractive libraries. Selected clones were also used to examine gene expression in crown buds after growth induction and/or during normal seasonal growth. In this study, four distinct patterns of gene expression were observed during the transition from para-dormancy to growth-induction. Many of the differentially regulated genes identified have unknown or hypothetical functions while others are known to play important roles in molecular functions. Gene ontology analysis identified a greater proportion of genes involved with catalytic activity in the forward library while the reverse library had a greater proportion of genes involved in DNA/RNA binding.