Immunolocalization Of Ubiquitin And Cysteine Protease In Laticifers Of Euphorbia Helioscopia L.

Euphorbia helioscopia L. belonging to Euphorbia (Euphorbiaceae) is an annual or biannual herbaceous species, and the whole plant is used as a folk medicine. It has also been applied in clinical medicine now. The laticifers of E. helioswpia is a nonarticulated type and with some branches during tissue development, it is distributed throughout the whole plant. Laticifers contain white latex which is mostly the secondary metabolites of plant and has the effect of protection. The composition of latex is complicated and has the medicinal value. In our former works, the degradation of protoplast during the development of laticifers in E. helioswpia was observed with electronic microscopy. With latex proteomics, some enzymes which involved in protein degradation in latex of E. helioswpia were detected, and the gene cloning, prokaryotic expression and antibodies preparing of some protease has been completed. This study mainly focused on the immunolocalization of ubiquitin and cysteine protease in laticifers of E. helioswpia with the methods of western blotting, immunocytochemistry labeling and colloidal gold immunoelectron microscopy. Such information can provide evidences for the further understanding of the development of laticifers. The main results were as follows:1. Through the experiment of western blot, it was found that ubiquitin antibodies labeled several stripes to the total latex protein, which showed that the latex contained ubiquitinated proteins and the ubiquitin-proteasome pathway may participated in the development of laticifers. Cysteine protease antibodies identified one stripe to the total latex protein, its relative molecular mass was similar with the expectation, and so it proved the availability of cysteine protease antibodies on one hand and the involvement of cysteine protease in the growth of laticifers on the other hand.2. Through the immunohistochemical observation with paraffin sectioning technique, the ubiquitin antigen signal in laticifers of E. helioswpia stems was detected. The development of laticifers is earlier than vascular bundles. The ubiquitin antigen signal was observed increasing gradually during the development of laticifers until it matured, which showed that ubiquitin was involved in the development of laticifers. While, the antigen signal in the cell around laticifers was also observed, but with the development of tissue, the ubiquitin antigen signal had a tendency to reduce.3. The subcellular localization of ubiquitin and cysteine protease in laticifers by using colloidal gold immunoelectron microscopy indicated that gold particles with ubiquitin antibodies were randomly distributed in the degenerating protoplasm. For instance, the gold particles were mostly distributed around the endoplasmic reticulum, dicytosomes and mitochondria or in the degenerating nucleus. The gold particles labeled by cysteine protease antibody were mainly distributed in the small vesicles of degenerating protoplasm, the gold particles were also found in the nucleus, endoplasmic reticulum, autophagosomes and it's nearby. As a result, ubiquitin was involved in the development of E. helioscopia laticifers and cysteine protease participated in the protoplasma degeneration of E. helioscopia laticifers through macroautophagy.