Identification Of Rice Varieties Against Ustilaginoidea Virens And Cloning As Well As Prokaryotic Expression Of Amidase Gene Of The Pathogen

Rice false smut, caused by Ustilaginoidea virens (Cooke.) Takahashi, is more serious with the cultivation of hybrid rice, especially super hybrid rice expanded and increased level of nitrogen as well as global warming in recent years. It has become one of the main fungal disease of rice. Rice false smut doesn’t only affect the rice yield, but also produce the toxins harmful to human and animals, serious damage to food security.The study mainly carries out the artificial inoculation of U. virens, using the electroporation method to gain the transgenic U.virens isolates and Cloning Amidase Gene of U.virens as well as Prokaryotic Expression to expect to improve the efficiency of the evaluation on resistance, to reveal the infection circulation of U.virens and lay a foundation to research the function of amidase gene in U.virens.1.54U.virens isolates were identified by using the primers of ITS4,ITS5to identify rDNA-ITS region from different sources, the resistant reaction of12rice varieties on11different sources of U.virens isolates were studied. The results showed that the rDNA-ITS region of U.virens was highly conservative. Some resistance differentiation was observed when the same rice variety has been inoculated with different U. virens isolates, while different rice varieties have been inoculated with the same U.virens isolates were do so. Moreover, the resistance diversity was also observed between the first season rice and the ratoon rice subjected to the same treatment.2.The plasmid (contained GFP gene and Hygromycin resistance gene) was transferred into thin-wall conidia using the electroporation. It can be seen green fluorescence sent out from transformational isolates when observed the mycelium of transgenic U.virens isolates under fluorescence microscope after being cultivated5to7days. DNA of transgenic U.virens isolates were extracted, amplified by using primers of Hygromycin primers, the result of agarose gel electrophoresis was corresponded with the expected result (1023bp). The result showed that U.virens transferred with GFP gene and Hygromycin resistance gene was gained.3.The total RNA of U.virens was extract by modified Trizol method. According to the registered sequence of Amidase of U.virens, Amidase gene of U.virens was successfully obtained by designing primers of ACER and ACEF using RT-PCR. Primers of RCP and FCP were redesigned to amplify amidase gene of U.virens, recombinant amidase gene was obtained. Recombinant amidase gene was connected to recombinant prokaryotic expression vector pQE30, then transformed into E.coli BL21and extracted extracellular protein. Through SDS-PAGE electrophoresis, coomassie brilliant blue staining and decoloration, the results showed that amidase gene was successfully expressed.