MUNCIE, INDIANA
Ball State University1
TOLEDO, OHIO
University of Toledo2
WOODWARD, OKLAHOMA
USDA-ARS-SPRRS3(posthumously, 10/02)
NOVOSIBIRSK, RUSSIA
Institute of Cytology and Genetics4
Isolation of stage- and ploidy-specific floral RNA in Tripsacum dactyloides for cDNA library construction - a pilot study for large-scale isolations --Blakey, CA1, Houghteling, B1, Goldman, SL2, Dewald, CL3, Sokolov, VA4, Hammersmith, R1 A pilot study was designed and conducted at Ball State Univ. and incorporated into part of a Master's thesis project (B. Houghteling, 1998, " Isolation of differentially expressed messages in sexually reproducing Tripsacum dactyloides." M.Sc., BSU.). The aim of the study was to determine the physical stages of floral development for the isolation of pre-emergent ovules of greenhouse plants for both WW1582 (sexual) and WW1008 (asexual, apomictic) in the range of 0.5 to 3.0 mm, and to develop the step-by-step procedure for harvesting tissues containing these ovules with minimal contamination for high quality RNA isolations, necessary for later large-scale isolations from field nurseries. The field total RNA isolations would be used in the creation of stage- and ploidy-specific cDNA libraries, therefore the preservation of the RNA, particularly rare mRNAs, was a primary goal of the project. Harvested pre-emergent spikes were sized and ovules were excised to determine an approximate physical correlation between fruitcase width and ovule width within the fruitcase. Based on these findings a set of pre-emergent spikes with fruitcases were divided into three classes, <0.5 mm (E), 1.0-3.0 mm (M), and > 3.5 mm (L) (all fruitcases used were less than 5 mm in width). Total RNA was isolated from these sample classes using a Trizol&trade; RNA isolation protocol (as provided by Sigma).

Stage-specific cDNAs were generated using the SMART&trade; PCR cDNA Synthesis Kit (CLONTECH Cat# K1052-1). Small quantities of cDNAs were detected for each class, but in insufficient quantities for library construction. In order to enhance total RNA isolations, larger quantities of staged tissues of these accessions would be required. Based on the work at Ball State, it was determined that the fruitcase width measurements would be altered to compensate for the higher quantities of advanced maturity sexual spikes relative to the availablity of early apomictic spikes during the limited RNA isolation experiment time-window in Oklahoma.

Racemes with maturing terminal inflorescences were harvested from multiple clones of the sexual diploid (2x=2n=36) Tripsacum dactyloides plant WW1582 from Ottawa, KS, and from multiple clones of the tetraploid (4n=4x=72) Tripsacum plant WW1008 from Baird, TX, by Dewald and crew in Woodward, OK. Lateral pre-emergent inflorescence spikelet sections were then isolated from these racemes. Proximal cross-sectional cuts were made immediately below the node of inflorescence development, and the distal cuts were made 5-7 cm above the node. Outer leaves were removed from these sections by lateral incisions on the side opposite the inflorescence structure. The final layer of immature leaves encasing the inflorescences was left intact to maintain tissue sterility.

The encased spikelet sections were placed on ice until the floral structure was removed and sized, under sterile conditions, to reduce RNA and/or RNase contamination of female floral tissues. The immature spikelet sections were size-sorted according to the width of the ovule fruitcase. The classifications were as follows: "E" for early stage (<1.5 mm), "M" for middle stage (1.5-3.5 mm), and "L" for late stage (>3.5 mm). Ovules were not excised from surrounding maternally-derived tissues (fruitcase and stem); thus, the entire floral section was used in subsequent RNA isolations. It is important to note that these sections did not contain any male floral structures. The actual Trizol&trade; RNA isolation was performed on-site in Woodward, OK, by Blakey, according to the Trizol&trade; RNA isolation protocol provided by Sigma. Total RNA was stored under 75% EtOH in DEPC-H2O at 20 C and transported to Ball State University (BSU), Muncie, IN, for quantitation. Following resuspension, the total RNA samples were quantitated by UV at 260/280 nm. Resuspended total RNA samples were stored at -80 C until samples were divided. A portion was placed in permanent storage at BSU. The remainder of the RNA was shipped to the PSRC facilities, University of Toledo for 5 of 6 cDNA library constructions, and residual U.Toledo RNA subsequently sent to Miami University, Oxford, OH, for suppression subtraction hybridization library construction.

Homology searches from sequenced cDNAs obtained from the U. Toledo libraries, to date, will continue to be analyzed at Ball State University. Update searches and re-analysis of novel clones will also be undertaken relative to any newly released Oryza, Arabidopsis, and Zea sequences will continue to be performed.
 
 
 
 


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