The Immuno-Cytochemistry On Pollen Tube Special Protein Of Citrus Grandis Var. Shatinyu Hort.

In this research, immunogold (GRA-Au complexs) electron microscopy cytochemistry techniques are used to study the localization of S-proteins and S₁-RNase in the pollen and pollen tube of 1/2 style site of Citrus grandis var. Shatinyu hort.3 days after self-pollination. At the same time, the better conditions for the pollen culture of Shatinyu was discussed. The sources and the course of transportation of S-proteins and S1-RNase of the pollen tube were analyzed. So here we provided some new data for GSI ( Gametophytic Self-incompatibility) about pollen and have done some basic wok to finally identify the production of S-gene of Shatinyu. In genetic, the self-incompatibility in higher plant is controlled by S-locus. Its recognition protein is an S-locus special glycoprotein(SLSG) (S-protein) that is a kind of major function protein controlling higher plants, self-incompatibility. The recognition protein used in this experiment is abstracted from the suppressed pollen tube of Shatinyu. It is a production of S-gene. The antibody of special protein of pollen tube and S1-RNase were made and pured according to the method of LongXiang Zhang ,etc. . At the same time we utilized LowicrylK4M to bury 1/2 style piece 3 days after self-pollination and anther. Then immunogold electron microscopy cytochemistry techniques were used to study the localization of S-proteins and S1-RNase in the pollen and pollen tube of the 1/2 style site of shatinyu 3 days after self-pollination. The result of the experiment show the best condition for the pollen of shatinyu is to be. 15%of the cane sugar, 0.02%MgSO₄, 0.01%KNO₃, 0. 03%Ca(NO₃)₂, 0.01% H₃BiO₄, 20% PEG₆₀₀₀, pH5. 6. The result of immunogold label show that the S-protein are distributed in the endoplasmic reticulum, cytoplasm and cell fiber wall. In the pollen, they are located in the pollen wall, especially in sprout hole and pollen wall nearby it. The samples of control are not labeled by gold particles. The problem that the protein is finally formed while the pollen is growing or from pollen while the pollen sprouts is not solved so far .Our experiment indicates that there is S–protein that is distributed in the pollen in florescence. So we think the S-protein is got from the pollen. Before the pollen sprouting they are near pollen hole, and then enter pollen tube when the pollen sprout. Finally they participate recognition response. The growing pollen of is suppressed indicate that Shatinyu S-protein to be high-efficient is for 1-3 days after self-pollination. The results of fluorescence microscope observation show that Shatinyu pollen tube stop growing at the style 1/2 site. The results of immune enzyme label and gold label of self-pollination style show that S-protein is formed by the endoplasmic reticulum, transported and arrived cell fiber wall finally. These results verify style S-gene expression location and time and that of pollen tube are at the same time. This keeps the same with research of Cornih ,etc.. Science of heredity research indicate self-incompatibility is controlled by specific gene.The growing of pollen tube is suppressed by the pistil which have the same equal gene. Our experiment verifies that the S-proteins in the pollen, the pollen tube and the style are controlled by the same gene in genetic. So self-incompatibility is caused. In construction, the results show 3 days after self-pollination the pollen tube are passing the angular domain among several canal cells. The canal cells wall keeps in touch closely with pollen tube wall, which offer condition for recognition response. So we can think S-protein is reach certain threshold value to accumulate outside the fibre as 1/2 style passway cell after pollinating , the pollen tube is growing to style 1/2 site at this moment. S-protein of style is shifted to the pollen tube and recognition response is produced. Theseresults verify the recognition position is at the wall of pollen tube and the canal cell 1/2 style from cell's level. The results of gold label of S1-RNase is very similar with that of S-protein. The samples of control are not labeled by gold particles. From the results we know S1-RNase participate recognition response properly. Meanwhile, this experiment shows some direct evidences. It is a reliable method that studies the recognition site and the relationship of recognition protein time and space.