Physiology And Molecular Biology Of APX In The Process Of Somatic Embryogenesis In Dimocarpus Longan Lour.

In this experiment, longan embryogenic callus was synchronized by controlling 2,4-D concentration and development time; then the dynamic changes of the biomacromolecules of synchronized embryogenic cultures at different developmental stages were assayed; and the activity of APX which was closely related to somatic embryogenesis was detected, and the APX activity of the embryogenic cultures grown at different temperatures was also assayed; furthermore, the RNA was extracted from longan embryogenic callus to clone cytosolic ascorbate peroxidase gene(APX) for probing into the functional mechanism of APX in plant somatic embryogenesis, which would provide the clue for studies of the molecular mechanism of somatic embryogenesis in plants. The main results were as follows:1. The synchronization of longan somatic embryogenesis and the dynamic changes of the biomacromoleculesThe synchronized embryogenic cultures at different developmental stages were obtained by controlling 2,4—D concentration and developmental time.There were two peaks of starch accumulation in the process of somatic embryogenesis. one was at the stage of embryogenic callus, the other was at the stage of globular embryoids; and a small raise appeared at the stage of torpedo-shaped embryoids, and the content of starch was slightly higher than that of heart-shaped embryoids and cotyledonary embryoids. With the development of embryos, the tendency of the changes of DNA, RNA and the total nucleic acids were approximately similar, the peak value was at the stage of cotyledonary embryoids, and the change of RNA was more remarkable than that of DNA and the total nucleic acids. The tendency of change of proteins almost took on S-shaped curve. The dynamic changes of those biomacromoleculesplayed an important role during somatic embryogenesis.2. The change of APX activity in the process of longan somatic embryogenesisAPX activity was detected to exist at all developmental stages of synchronized embryogenic cultures in this experiment; and furthermore, the APX activity of the cotyledonary embryoid was at the peak, and it should be the total of the activities of the different soluble APX isoenzymes. The expression of APX gene was not synchronous to the APX activity, so the increase of APX activity could not be regarded as the enhancement of the expression level. The activity of longan APX might be controlled by two ways: modulating through protein-protein interactions and modulating through transcription by transcriptional or post-transcriptional regulation.At the following stages of embryogenic cultures—embryogenic callus, globular embryoid, cotyledonary embryoid, the APX activity was tested at 20 "C, 25 °C and 30 "C, respectively. The APX activity of the embryogenic cultures grown at 20 "C and 30 'C were compared with that of 25 "C, the difference between the activity at 20'C and the activity at 25 "C was not significant. The activity at 25"C was slightly higher than that at 20"C. The total activity at 30°C was approximately 1.5 times of that of which grown at 20°C. To some extent, high temperature could induce the expression of APX. Heat stress could trigger the antioxidant pathway to protection of common cellular functions which was linked to APX. This inducement was specific to different isoenzymes. At the early developmental stage of longan somatic embryogenesis, the mRNA of APX was detected at the normal temperature, and the APX activity was also tested, which concluded that there was the active APX expression at the satge, otherwise there was passive APX expression in response to biotic and abiotic stresses.3. The homologous cloning of longan cytosolic ascorbate peroxidase genePartial cytosolic ascorbate peroxidase gene (cAPX) was amplified and cloned from Dimocarpus longan embryogenic callus using 3' RACE (GenBank...