| Fusarium head blight or corn scab caused by Fusarium graminearum,not only led to yield losses,it produces a variety of toxins, include NIV and DON, threating to the health of human and animals. Currently, using of azoles pesticide, eg: carbendazim, is the main methods to controlling Fusarium head blight. Certain effects have achieved, but as years passed, the control methods have become increasingly prominent drawbacks. Firstly, pesticide pollution become more and more serious; secondly, prevention effect is getting worse because of constant emergence of drug-resistant strains. Therefore, the establishment of new methods and systems to prevent and control the disease is imminent.In this dissertation, RNAi technique was being used to study the effects of different fragments, which derived from Fg Pkc, the key gene of Fusarium asiaticum. The CDS sequence was divided into 8 fragments, 500 base pairs of each, constructed into the RNAi vectors. After identification by general and specific PCR, southern blot,10 mutants were obtained by protoplast transformation. 3rd and 5th got 2,respectively;the rest of fragments got 1 mutant each.To further research the effect of each fragments, the following experiments were done:Phenotypic test:cultured in SNA medium for 4 days. then determine colony area. The growth rate of Pkc3-3 had significant difference with the wild strain, the remaining mutants had extremely significant difference with wild. Compared to wild type, the growth rate of Pkc5-2 and Pkc5-12 has dropped by more than 60%; and Pkc3-3, only 4.88%. Pkc8-16 has no different with the wild type.Field inoculation: the results of pathogenicity show that there is no difference between wild strains and Pkc7-15, but Pkc4-7, Pkc5-2, Pkc5-12 and Pkc6-9 were significantly reduced. In addition, degree of pathogenicity caused by Pkc4-7, Pkc5-2, Pkc5-12 were more slight compared with others mutants. Compared to wild type, the pathogenicity of Pkc4-7, Pkc5-2, Pkc5-12 decreased by 72.10%, 57.12%, 54.96%, respectively.Coleoptile inoculation: the length of scab created by transformants shorter than the wild strain. The result show that Pkc5-2, Pkc5-12 lose part of virulence, the pathogenicity of this mutants dropped by 60.75%, 52.91%, compare with wild type. This consistent with the results of field inoculation.Conidia size: determination of each transformant and wild strain conidia length and width with microscope, to analyze the differences between them. All the mutants are smaller than the wild strain in length, and have no evident difference in width except for Pkc5-2.In summary, Fg Pkc is a important gene involved in growth, pathogenicity, reproduction in the life cycle of Fusarium asiaticum. Each fragment of the gene silenced by si RNA, the mutants shows significant difference with wild strain in gowth rate, field inoculation, coleoptile inoculation and the size of conidia. Particularly, the transformants of the 5th fragment, show more obvious difference when compared with others and the wild strain. And there are two independent mutants fo the 5th, this may provide a strong evidence that the 5th fragment of Fg Pkc play a key role in the life cycle of Fusarium asiaticum. Construction of plant expression vector include the 5th fragment, si RNA may inhibit the growth of Fusarium asiaticum, the disease may be in control. |
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