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United States Department of Agriculture

Agricultural Research Service

Research Project: SUCROSE ACCUMULATION AND RETENTION IN SUGARBEETS

Location: Sugarbeet and Potato Research

Title: In situ hybridization of riboprobes for two sucrose synthase transcripts in sugarbeet root tissue

Authors
item Martins, Daniel - MINISTERIO DA AGRICLTURA
item Fugate, Karen
item Finger, Fernando - UNIVERSIDADE FEDERAL VICO

Submitted to: Brazilian Congress of Plant Physiology
Publication Type: Abstract Only
Publication Acceptance Date: July 20, 2007
Publication Date: September 1, 2007
Citation: Martins, D.N., Klotz, K.L., Finger, F.L. 2007. In situ hybridization of riboprobes for two sucrose synthase transcripts in sugarbeet root tissue [abstract.] Brazilian Journal of Plant Physiology. 19(Supplement).

Technical Abstract: Sucrose synthase is an important enzyme for sucrose metabolism in sugarbeet (Beta vulgaris L.) roots. Its activity rises during root development and it is correlated with sucrose accumulation. Sucrose synthase has two active isoforms in sugarbeet roots. The goals of this work were to study spatial distribution of sucrose synthase mRNA in young sugarbeet roots. RNA was isolated from roots, and RT-PCR with specific primers for each sucrose synthase gene was performed to produce DNA probes. DNA probes were used in in vitro transcription reactions to produce DIG (digoxigenin) labeled RNA probes in sense and anti-sense direction for both genes. Specificity of each probe was verified by hybridization with mRNA of SBSS1 and SBSS2 genes fixed on charged membranes in dot blot hybridization assays. For in situ hybridization, 6 week old tissue of sugarbeet root was fixed with FAA for 12 hours at 4 ºC. The tissue was embedded in Paraplast, sectioned with a microtome and fixed on slides. Tissue on slides was deparaffinized with Histoclear and hydrated in a series of decreasing concentration of ethanol. Then, it was treated with proteinase K, followed by tissue refixation with 4% paraformaldehyde and acetylation treatment. Afterwards, tissue was dehydrated in a reverse series of ethanol rinses. Separate slides with tissue were used for the hybridization reaction with each labeled probe, sense and antisense. Hybridization was performed at 55 ºC for 18 hours. After hybridization, tissue was subjected to high stringency washing and RNase treatment. Immunohistochemical assay was performed with anti-DIG antibody conjugated with alkaline phosphatase. Final color was developed by incubation with NBT/BCIP substrate. Transcripts of SBSS1 and SBSS2 genes did not differ in relation to localization in tissue. They were found mainly in storage parenchyma. No sucrose synthase mRNA was detected in growth tissues or in vascular bundles. The results suggest that parenchyma develops a central role in sucrose storage and distribution, and sucrose synthase may be involved in the regulation of these processes.

Last Modified: 8/29/2014
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