| Helminthosporium gramineum Rabenh f. sp. echinochloae (HGE), isolated from diseased barnyardgrass plants in Nanning, Guangxi province by this research group. We have found that the fungal pathogen, HGE can cause severe poisoning and even killing effect on weeds in rice fields and be safe to the rice. Also, HGE could produce some pathotoxins, such as Ophiobolin A. These toxins had the lethal function on weeds, so they can be developed as mycoherbicidal agents for the control of barnyardgrass and other weeds in rice fields. Restriction enzyme mediated integration (REMI) was firstly used in Saccharomyces cerevisiae integration research. At present, this technology is generally utilized in the practice of transformation of fungi and gene mark. In this paper, the objective is to use plasmid pSH75 to transform HGE to get mutants which are not able to produce ophiobolin A, as well as to form a base of the following studies on the mutants to find out the functional genes related with Ophiobolin A and know its metabolic pathway. The main contents of my dissertation are as follows:Formation and regeneration protoplasts of HGE. HGE,one species of filamentous fungi, has complicated cell wall structure to prevent the cells from foreign plasmids. So it is a key step to establish an effective system of preparation and regeneration of HGE protoplasts. The best conditions for isolating protoplasts are that digesting the 6-hour mycelium for two hours at 26℃by 0.5%Drislase+0.5%Lyase+0.5%Snailase in 0.4mol/L MgSO4 solution. Otherwise, the most regenerated colonies of HGE protoplasts have been harvested under the conditions that lysing the mycelium which was cultivated 16 hours with 0.5% Drislase in 0.7 mol/L NaCl at 32℃. The lysing system of formation differs from that of regeneration of protoplasts of HGE. However, it is possible to increase the gross of proplasts through digesting more mycelium. To acquire enough regenerated colonies for following experiments, we choose the second situtation as the optimum conditions. Some other important factors impacting on regenerated rate have also been tested.Transformation of HGE protoplasts and screening of the mutants. The plasmid pSH75 was inserted into the genome of HGE at random, and after regenertiaon of the protoplasts, a number of mutants were harvested on the selective medium with 40μg/mL hygromycin B. After screening, it was abandoned that the aborted transformants and the slow-growing mutants which will not be accustomed to the next experiments. It was used to identify the target one that testing the inhibition of rice sheath blight, biocontrol of barnyardgrass in greenhouse treated with the transformants, and detection of ophiobolin A produced by the mutants. What's more, we synthesised two pairs of primers to PCR the amp and hph genes in these mutational genomes. The results indicated that the plasmid pSH75 had been inserted into the fungus genomes. It was confirmed that NO.B014 transformant is Ophiobolin A deficit mutant.Finally, it was studied that the inhibition of rice bacterial leaf stripe treated with the filtrate and crude toxins of HGE. The filtrate and crude toxins (ethyl acetate extract of culture filtrate of HGE) of HGE were tested with the method of stiletto, and measured in MIC (Mininum Inhibitory Concentration) and MBC (Minimal Bacteriocidal Concentration), compared with the untreated checks of 20% thiodiazole-copper, 72% streptomycin sulfate and bismerthlazol. The results indicated that the inhibitory effect from the treatment of the crude toxins of HGE was much better than other treatments.
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