Subcellular Localization Of α-galactosidases In Cucumber (Cucumis Sativus L.) By Green Fluorescent Protein Fusion Expression

Cucumber(Cucumis sativus L.) primarily transports stachyose in the phloem, while in the fruit, the predominant soluble sugars are sucrose, glucose and fructose, little raffinose family oligosaccharides can be detected in this tissue. Nevertheless, the details how stachyose is unloaded from the phloem and then converted to sucrose still remains poorly understood by far. Investigating the space expression pattern at subcellular level ofα-galactosidase (EC 3.2.1.22), a key enzyme catalyzing stachyose catabolism, is beneficial to answer these questions.Therefor,two kinds of cucumberα-galactosidase-EGFP fusion expression vector controlled by the CaMV 35S promoter were constructed. CaMV 35S promoter sequence was amplified by polymerase chain reactions (PCR) from vector pCambia 1303 ; The entire coding region of EGFP was amplified by PCR from vector pEGFP-N1;The entire coding region of cucumber acidα-galactosidaseⅠand alkalineα-galactosidaseⅠwere amplified by Reverse transcript PCR using cucumber leaf total RNAs as template. The three above-mentioned fragments were Inserted into the multiple cloning sites of expression vector pCambia 1381*c.Then twoα-galactosidase-EGFP fusion expression vectors were constructed respectively and the EGFP gene was located at the C- terminal of the target gene.Finally, the two recombinant plasmids that were transformed into onion epidermal cells and cucumber callus cells by particle bombardment. Green fluorescence signals was monitored by a confocal microscope.The results showed cucumber acidα-galactosidaseⅠis located in the whole cells includeing the vacuole, while cucumber alkalineα-galactosidaseⅠis located at the nucleus and the peripheral region of the cell.