| In this study we discussed the better conditions for the pollen culture of Citrus grandis var. Shatinyu Hort. in vitro and made some research on the method of ultrasonic waves on extracting proteins from the pollen tube．In addition,we established the two-dimensional electrophoresis analysis technology that is highly sensitive and demands less loading．Using the two-dimensional electrophoresis together with silver staining, we compared and analyzed the proteins extracted from self and non-self pollinated pollen tube．Moreover,the types and activity of three kinds of isoenzymes of Shatinyu pollen,i.e. peroxidase(POD),amylase(Amyl)and esterase(Est)were analyzed with vertical slab PAGE and by specific cytochemical staining during the process of pollen germination．So here we provided some new data for GSI(Gametophytic Self Incompatibility)about pollen and have done some basic work to finally identify the male determinate of GSI in Shatinyu．Pollen that has been stored at -70℃ was used in the experiments to determine the time course of pollen tube growth,the effects of pollen density on germination rate of pollen and the effects of osmotic potential of the medium on pollen tube growth．Finding that it is most important considering the moisture content of pollen,the temperature under storing and the density of pollen when sowed in the culture medium．We got the highest germination rate about 80ï¼… to 90ï¼… when meeting the needs such as the moisture content of pollen ranges from 11.0% to 12.5% during the culture of pollen tube．To maximize pollen tube growth,we also optimized pollen density and the concentration of sucrose．The medium contained 12% sucrose is better though the pollen is to be well germinated under 10% to 20% sucrose．The density of pollen required to achieve the highest growth rate was 8 to 12 anthers' pollen in concave utensil(?5ãŽ) with 1.5ml culture medium or 6 to 8 anthers' pollen in microscope slides with 0.5ml culture medium．Both the concave utensil and the microscope were incubated in the dark in the plate with wet paper at 28℃ to 30℃．Measured the extracts by SDS-PAGE after the pollen tubes were processed by ultrasonic waves．Compared with the routine method,the efficiency of extracting proteins from pollen tubes was greatly enhanced and the result displays more stable and dependable by ultrasonic process．So proteins extracted by ultrasonic process is significantly higher than that extracted by the routine method．We did the experiment with a series of parameters to find out which were better for the result．After having done 120 times as ultrasonic waves lasts 3 seconds every 9 seconds under 600W power,90 percent pollen tubes were crashed into shatters but the microspores were almost integrated．Two specific proteins with molecular weights of 22.0ku and 32.5ku were detected in self-pollinated pollen tube while another specific protein about 44.8ku was less than the protein owing the same Mr in cross-pollinated pollen tube by SDS-PAGE．In addition,there were two bands whose pI are 5.85 and 6.20 found in self-pollinated pollen tube by the method of IEF-PAGE．Compared and analyzed the patterns of pollen tube proteins between self and non-self pollinated in the two-dimensional electrophoresis,their proteins' distribution is similar and shows overlapping．More than 200 spots are differentiated on the gel．Two special proteins spots were detected in self-pollinated pollen tube electrophoresis．Their molecular weights are 28.0ku and 26.4ku,respectively．Their pIs are 6.30 and 6.10 approximately．However,a 58.2ku protein whose pI is about 5.90 was different from self-pollinated pollen tube in the two-dimensional electrophoresis of cross-pollinated．These three kinds of proteins from pollen tube of Shatinyu maybe interrelate to self-incompatibility．Herein the two hypothesis about GSI,our result supports the RNase inhibitor model rather than the receptor or gatekeeper model on the one hand．Compared the types and the activity of three kinds of isoenzymes of Shtinyu germ... |
