Constructed Obsenvation And Subcellular Localization Of Calcium Of Wheat Near Isogenic Line TcLr19 Under The Stress Of Puccinia Triticina

Wheat NIL TcLr19 appeared durable and high perfermance resistance to leaf rust fungus for a few of this fungus species possess virulence to it, but the physiological functions of its excellent resistance are not well understood. TcLr19 was focused on by the stablity and efficiency of its resistance to leaf rust,whereas the excellent mechanism was unrevealed. Cytobiology study on the resistant merchanism would prove finding and cloning of important founctional resistant genes, reveal moleculer regulation mechanism of wheat resistance to leaf rust disease, promote new heavy producing resistant wheat species breeding.In this study, photology micrological technique and transmission electron microscope were used to observe micrological structure and ultramicrostructure of wheat mesophyll cell structure during the different combination interactions, the compatibale combination of NIL TcLr19-leaf rust fungus species 366 and incompatible combination Tc-366. And then, biochemical characters of host nucleus DNA in both interaction combinations were systematic analysised by agarose gel electrophoresis. At last, the subcellular of distribution calciumion in early stage of interactions was located by calcium pyroantimonite method. Result showed as below:1. According to the observation result of micrological structure, in the incompatible combination, mycelial grew slowly, small amount mycelial exised. Wheat mesophyll cell structure changed 8 hpi, major manifestations were chlorophyll body and nuclear. Chlorophyll body location at changed, both organelles structures variated until vanished at last. At 96 hpi, the mesophyll cells near mycelial appearared irregularitible shape, maybe be going to death. However, in the compatible combination, mycelial grew fast, host cellstructures had little change, individual chlorophyll body changed its shape or location. It is means that invasion of mycelian did not affect host cell structure obviouly.2. Observation result of SEM revealed that: in the incompatible combination ,at 4 hpi, the irregular of nuclei became visible, in the early stage of inoculation , 4 hpi to 16 hpi, the chromatin was in agglutination and stayed at the edge of nuclei.The chloroplast became swelling, and the internal structure of chloroplast was in great disorder. Some mitochondria started to degrade at 16 hpi. In anaphase (24 hpi to 96 hpi), the nuclei and the chloroplast were completely disrupted. Mitochondrial membrane were normal, but its internal structure had disappeared. Vesicle and autophagic vacuole did play a vital role in the further degeneration of other organelles just similar to the lysosome acting in the animal cells. The cellular membrane retained integrity all the time. These results suggest that the ultrastructural feature of cell death was a form of PCD. In the compatible combination since 4 hpi to 16 hpi, the ultrastructure of host cell had no obvious change. Since 24 hpi to 48 hpi, the chloroplast became swelling, and the internal structure of chloroplast were in disorder,the volume and number of starch grain was increase. Since 72 hpi to 96 hpi, the chloroplast had varied shape, the alinement of basal granule was in surface loosening, the number of starch grain was increase, some mitochondria started to degrade, but the nuclei was normal.3. DNA ladder was detedcted from host leaves in incompatible combination since 8 hpi. In additional of that, DNA ladder was obtained from uninoculated leaves, leaves in compatible combination and leaf rust gungus spore. This result provided biochemistry proof to the hypothesis that the cell death in incompatible combination belong to PCD.4. The calcium ion variation in this system was: Ca2+ signal changed from 2 hip, Ca2+ inflowed from extracecullar to periplast, and at 8dpi, calcium deposition increase markedly in cytoplasm, in the compatible combination, and the increase of Ca2+ was delayed and unremarkable.The results showed above suggested that: After inoculated with leaf rust species 366, the HR-PCD of high resistance NIL TcLr19 occurred at 4h, Ca2+ involed in this process as second messager, and, its concentration increased at 2hpi.. Compared with other wheat-leaf rust fungus incompatible combination in our lab, L10-leaf rust fungus, the emerge time of Ca2+ signal was 6h earlier, and start time of HR-PCD was 8h earlier. We deduced that it might be the resistance cytobiology merchanism of TcLr19, and discussed about related studies in this thesis.