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Title: IDENTIFICATION OF DIFFERENTIALLY-EXPRESSED GENES IN DORMANT AND GROWING BUDS OF LEAFY SPURGE (EUPHORBIA ESULA L.)

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

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 7/10/2005
Publication Date: 7/16/2005
Citation: Jia, Y., Gu, Y.Q., Horvath, D.P., Anderson, J.V., Chao, W.S. 2005. Identification of differentially-expressed genes in dormant and growing buds of leafy spurge (Euphorbia esula L.). [Abstract]. Annual Meeting of the American Society of Plant Biologists. Final Program and Abstract Supplement, Poster 670, Page 225.

Interpretive Summary: Leafy spurge is an invasive perennial weed that infests range and recreational lands in the Northern Great Plains. Dormancy in these buds contributes to persistence of leafy spurge and makes current control methods difficult. These buds develop during the normal growing season but are maintained in a quiescent state through correlative inhibition. In the fall, these buds develop innate dormancy prior to over wintering. To gain a better understanding of bud dormancy and growth in leafy spurge, we developed two subtracted cDNA libraries to search for differentially-expressed genes in dormant or growing buds. To identify individual clones represented in the libraries, 15,744 clones from both libraries were spotted onto membranes and screened several times by hybridizion with radioactive probes generated from different pools of cDNA clones known to be redundant. Eventually, 2,304 non-hybridizing clones were sequenced and found to represent 509 individual clones. DNA inserts from the 509 individual clones were spotted onto membrane for macroarray analysis. Radioactive probes were made from RNAs extracted from crown buds of intact (control) and a series of induced (2 hr, 2 day, and 4 day after decapitation) plants. Macroarry analyses identified 51 differentially-expressed clones. Semi quantitative RT-PCR was used to confirm macroarray results. RT-PCR was also used to monitor expression of other transcripts that were not sufficiently expressed in the buds to allow detection by hybridization. Many of the genes identified were differentially-regulated. Some of these genes are known to play important roles in molecular functions such as catalytic activity, transporter activity, while others are hormone-regulated or still have unknown or hypothetical functions.

Technical Abstract: Leafy spurge is an invasive perennial weed that proliferates from an abundance of underground adventitious buds. Dormancy in these buds contributes to persistence of leafy spurge and makes current control methods difficult. To identify genetic mechanisms regulating dormancy status, we developed two subtracted cDNA libraries (forward and reverse). Random sequencing of 100 cDNA clones from each library revealed that both libraries contained many redundant clones. To select non-redundant clones, 15744 clones from both libraries were spotted onto membranes and screened with clones known to be highly redundant. After many rounds of screening, a total of 509 unique sequences were obtained after sequencing 2304 non-hybridizing clones. DNA inserts from the 509 individual clones were spotted onto membrane for macroarray analysis. Radioactive probes developed from RNAs extracted from crown buds of either intact (control) or a series of growth induced (2h, 2d, and 4d after decapitation) plants were used to identify differentially-expressed candidate genes. Macroarry analyses identified 51 clones whose hybridization intensity in growth-induced samples was above or below 1.5 fold of the 0 hour control. Semi-quantitative RT-PCR was used to confirm macroarray results and to determine the expression profiles for other transcripts that are not abundantly expressed in root buds. RT-PCR was also used to determine the level of mRNA expression in crown buds after growth induction and/or during normal seasonal growth. Many of the genes identified were differentially-regulated. Some of these genes are known to play important roles in molecular functions such as catalytic activity, transporter activity, while others are hormone-regulated or still have unknown or hypothetical functions.