Abstract：This study is to supply a series of vectors for gene knockout, overexpression, and expressing fluorescent fusion proteins in the rice blast fungus. These vectors should be easily worked, time-saving, reliable, and conveniently for the research in the gene function of Magnaporthe oryzae. PCR, enzyme digestion, ligation and transformation were used to construct the plasmids．We cloned two promoters (SOD1 promoter and H3 promoter) which strongly expressed in the mycelia and conidia, built 3 vectors for knockout (pBS-SUR, pBS-BAS, pBS-NEO), 4 vectors for overexpression (pKD5, pKD6, pKD61 and pKD8), and 7 vectors for fluorescent fusion protein expression (pKD5-GFP, pKD5-RED, pKD6-GFP, pKD6-RED, pKD7-RED, pKD8-GFP and pKD8-RED)in M. oryzae and other filamentous fungi. These plasmids could be introduced into the fungi via protoplast transformation, or Agrobacterium tumefaciens-mediated transformation. The knockout mutants could be identified by PCR, Southern blot and Western blot, the fluorescence of the transformants was observed under the fluorescent microscope, and the expression level of genes in the transformants was assayed by Real-time PCR. We have used knockout constructs built by these knockout vectors (pBS-SUR, pBS-BAS, pBS-NEO) and pBS-HPH1 together to knockout 4 genes simultaneously in M. oryzae. After transforming pKD5-RED, pKD6-GFP or pKD6-RED into M. oryzae via Agrobacterium tumefaciens-mediated transformation, green or red fluorescent fusion proteins under the control of SOD1 or H3 promoter were expressed strongly in the hyphae; Real-time PCR results showed eGFP mRNA or DsRED2 mRNA level promoted by SOD1 promoter was 2.5 or 5.4 folds of β-tubulin, and DsRED2 mRNA level promoted by H3 promoter was 20.8 folds of β-tubulin in the mycelia. MoATG8-DsRED2 fusion protein produced by pKD6-RED could locate MoATG8 exactly in the nearby of vacuoles. Observation on DsRED2 fluorescent protein under microscope showed that DsRED2 under control of SOD1 promoter could express in several stages of M. oryzae, such as hypha, conidium and appressorium. These facts imply that above-mentioned 14 vectors can be used to knockout genes, overexpress genes, and express fluorescent fusion proteins in the rice blast fungus and other filamentous fungi, such as Fusarium.
