In Situ Localization Of 2 Alkali α-galactosidase MRNAs In Cucumber Peduncles (Cucumis Sativus L.)

Stachyose is the major assimilate translocated in the cucumber (Cucumis sativus L.) phloem. While in the fruit, the predominant soluble sugars are sucrose, glucose and fructose, little raffinose family oligosaccharides can be detected in this tissue. Investigating the space expression pattern ofα-galactosidase (EC3.2.1.22), a key enzyme catalyzing stachyose catabolism, is necessary to illustrate the stachyose unloading mechanism in cucumber. In this study, in situ hybridization was used to study the cellular localization of two alkaliα-galactosidase mRNAs in cucumber peduncles by digoxigenin-labelled RNA probes.Preliminary experiments of paraffin section showed clearly bicollateral vascular bundle structures in the cucumber peduncle, and 10μm cross-sections were selected for further in situ hybridization analyisis of this tissue.Accroding to the sequences of two alkaliα-galactosidase cDNAs, gene specific primers were designed and non-homological cDNA fragments were amplified by Reverse Transcription- Polymerase Chain Reacion(RT-PCR). The cDNA fragments were engineered into the T-vector pGEM-TE, which contain RNA polymerase promoters T7 and SP6. Digoxigenin-labelled RNA probes were synthesized by in virto transciption using these vectors as template. High labelled and hybridization efficiency of these probes were confirmed by dot-blots.In situ hybridization showed that both alkaliα-galactosidase mRNAs are expressed in the phloem and cortex cells in the cucumber peduncle. mRNAs of alkaliα-galactosidaseⅡis expressed more strongly than that of alkaliα-galactosidaseⅠin the phloem. The results indicated that phloem may be an important site of stachyose unloading in the cucumber peduncles.