The Differences Between Products Of Gene Expression In Male, Female And Hermaphrodite Cucumber Floral Buds (Cucumis Sativus L.)

Cucumber is a species in which sex expression has been extensively studied. Sexual differentiation is controlled by genotypic and environmental factors. The main genes responsible for sex determination have been described but the mechanism of their action remains unexplained. In this study we attempted to find cDNA clones which can be connected with sex differentiation and flower development in cucumber. 
Two pairs of nearly isogenic lines: GY-3 (gynoecious – FFMMGG) and HGY-3 (hermaphrodite - FFmmGG), B10 (monoecious - ffMMGG) and 2gg (gynoecious - FFMMgg) were used to search for differences in gene expression in young (1 – 2mm) cucumber floral buds.
In order to obtain differentially expressed cDNA clones the differential screening and the differential subtraction chain (DSC) methods were used. Altogether above 900 cDNA clones were isolated and part of them were randomly chosen and sequenced (tab. 1 and 2). 
To observe the expression patterns of isolated cDNA clones in developing flowers at different developmental stages, we performed in situ RT-PCR. Here we present the results for two cDNA clones designed as 216GY3 and 35GY3.The expression of 35 Gy3 clone, similar to hypotehetical protein from A. Thaliana, was localised in the stamen primordium and petals of floral buds, whereas there was no expression in male and hermaphrodite floral buds. The 216 Gy3 cDNA clone similar to chaperonine 60 beta chain precursor was strongly expressed in the pistil primordium in the male buds, whereas in female and hermaphrodite buds the oserved signals was very weak.

SUMMARY & CONCLUSIONS
The most of isolated and identified clones are involved in same part of a light or hormone signaling cascade and they may participate in the “cascade of sex expression” in cucumber. 
Signals of expression of clone 35GY3 were observed only in female cucumber flower buds and were the strongest in a site where as it seems only the development of stamens should be inhibited. 
The accumulation of large amounts of the transcripts of 216 GY3 clone in primordia of male flower pistils which will thus not develop is interesting.
 

References
  1. Malepszy S, Niemirowicz – Szczytt K., 1991. Sex determination in cucumber (Cucumis sativus L.) as a model system for molecular biology. Plant science, 80: 39-47
 2. LuoJ., Puc J.A., Slosberg E.D., Yao Y., Bruce J.N., Wright T.C., Becich M.J., Parsons R., 1999. Differential Subtraction chain, a method for identyfing differebces in genomic DNA and mRNA. Nucleic Acid research, vol, 27, No.19: e24.
 3. Urbańczyk-Wochniak, E., Filipecki, M., Przybecki, Z. A useful protocol for in situ RT-PCR on plant tissues. Cellular & Molecular Biology Letters 7 (1) (2002) 7-18. .
 4. Przybecki Z., M.E. Kowalczyk, E. Siedlecka, E. Urbanczyk-Wochniak, S. Malepszy, 2003. The isolation of cDNA clones from cucumber (Cucumis sativus L.) floral buds coming from plants differing in sex. Cell. Mol. Biol. Lett vol. 8, 421-438
 5. Przybecki Z., Kowalczyk M.E., Witkowicz J., Filipecki M., Siedlecka E. 2004. Polymorphom of sexually different cucumber (Cucumis sativus L.) NIL lines. Cell. Mol. Biol. Lett 9 (4B), 919-933.

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