关键词: 玉米, 拟轮枝镰孢, 宿主诱导基因沉默, 转基因, 穗粒腐病

Abstract:

【Objective】Fusarium verticillioides (F. verticillioides) is a common pathogen, which can cause ear rot, stalk rot, seedling blight, and seed rot in maize. These diseases caused by Fusarium verticillioides not only affected the yield and quality of maize, but also seriously threatened to the safety of human and livestock by a variety of fungal toxins such as fumonisin which produced during the metabolic process of the pathogen. So far, there is no report about the major resistance gene cloned and utilized for Fusarium verticillioides in maize. Using host-induced gene silencing technology provides a new strategy for resistance breeding in maize. 【Method】Key genes associated with the Fusarium verticillioides development were cloned using homologous gene sequence method, and the dsRNA were produced by in-vitro transcription (IVT) assay. The dsRNA for different genes was premixed with suspension spores of Fusarium verticillioides used for RNA silencing experiment in vitro. For investigate the degree of disease, the seeds of the susceptible inbred line Xi502 were sterilized and inoculated, and then were cultured in a petri dish at 28℃ in the dark for 48 h. For investigate the incidence of the seeds after inoculation, glucose was added to the spore suspension mixed with dsRNA, then spore germination and mycelia growth were observed under the microscope after 25℃ culture for 24 h. The Xi502 seedlings of the trifoliate stage were transferred to the spore suspension with premixed dsRNA for culture, and the incidence of seedlings blight was observed after 7 days. In order to select the target gene for HIGS, combine the seed morphological observation result after inoculation and seedling inoculation result. Then, the silent vector about these key target genes were constructed and transferred into the susceptible inbred line Xi502. The transgenic seeds were evaluated by artificial inoculation. The total RNA of the transgenic seeds after inoculation was extracted, and the relative expression of target genes in F. verticillioide was analyzed by qRT-PCR to determine the silencing effect of HIGS line. 【Result】Eighteen candidate genes related to growth were cloned by homologous cloning method in Fusarium verticillioides. It was found that the disease level of seeds was significantly reduced after 11 candidate genes silencing by seed inoculation experiments. Furthermore, six of the 11 candidate target genes, deo, Ras2, Dpdc, Hsp90, Frp1, and Atg15, were found that response to the spore germination and mycelium growth after gene silencing. Finally, based on the results of seedling inoculation, 3 silencing target genes deo, Atg15 and Frp1 with significant inhibitory effect in vitro were selected. Then the silencing vector was constructed by combine three specific segments from the three target genes, transgenic plants were obtained. It was found that the resistance level was highly increased in T2-generation seeds compared to the none-transgenic plants. As well as the expression levels of all the three target genes were significantly decreased in Fusarium verticillioides. 【Conclusion】Three genes, deo, Atg15 and Frp1, are important for development of Fusarium verticillioides. By constructing transgenic HIGS plants for target gene deo, Atg15 and Frp1, the increase the resistance to Fusarium verticillioides in maize.

Key words: maize (Zea mays L.), Fusarium verticillioides, host-induced gene silencing, transgenic, ear rot