Botrytis cinerea genes Bcmet3 and Bcmet16 related to pathogenicity and application
A technology of Botrytis cinerea and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc.
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Embodiment 1
[0027] Example 1 Construction and gene complementation of the sulfur-metabolism-deficient strains ΔBcmet3 and ΔBcmet16 of Botrytis cinerea
[0028] 1.1 Knockout vector construction
[0029] Bcmet3 gene (nucleotide sequence shown in SEQ ID NO: 1) and Bcmet16 gene (nucleotide sequence shown in SEQ ID NO: 2) are two key genes on the pathway of Botrytis cinerea assimilating external inorganic sulfur (sulfate ion) In this experiment, these two genes were silenced to explore the effect of sulfur assimilation on the fungus infecting fruit.
[0030] In this case, a transformation fragment was constructed by homologous recombination, and hygromycin phosphotransferase (HPH) was used as a selection marker. like figure 1 As shown, taking the Bcmet3 (Bcmet16) gene as an example, first, utilize the primer pair P1 / P2 and P3 / P4 to amplify from the wild-type strain B05. ) side fragment (see Table 1 for primers). Subsequently, a 1764 bp HPH cassette (hygromycin B resistant) containing the t...
Embodiment 2
[0040] Example 2 ΔBcmet3 and ΔBcmet16 Botrytis cinerea to spore germination and hyphal growth
[0041] 2.1 Experimental materials
[0042] 1) Pathogens tested: Botrytis cinerea (B05.10) was preserved and used in our laboratory, ΔBcmet3 and ΔBcmet16 were obtained from Example 1, and were cultured with PDA. The spores used in this case were cultivated on PDA medium within four generations .
[0043] 2) Test agent: PDB medium (potato dextrose broth medium, American BD company)
[0044] 2.2 Experimental method
[0045] Use a scraper to scrape mature fresh plate spores (cultured in PDA medium for 10-12 days), then rinse with 2 mL of 0.01% Triton X-100 sterile aqueous solution, pour the washed spore liquid into a 15 mL centrifuge tube, and repeat Rinse twice, pour all the obtained bacterial solution into a centrifuge tube, vortex and shake at 26°C for 5 minutes, filter with absorbent cotton to obtain a spore suspension, use a hemocytometer to count gray mold spores, and dilute to...
Embodiment 3
[0053] Example 3 Experiment of ΔBcmet3 and ΔBcmet16 Botrytis cinerea infecting fruit
[0054] 3.1 Experimental materials
[0055] Tomato fruit, cherry tomato "New Sun" (Solanum lycopersicum L.cv XinTaiyang), was freshly picked in the local field, 80% ripe, moved to the laboratory within 3 hours after picking, the fruit was soaked in 0.5% sodium hypochlorite for 4 minutes to sterilize the surface, and then Soak it in clean water for 2 minutes, wash it twice, and let it dry in the natural wind before use.
[0056] Gray mold strain (Botrytis cinerea, B05.10) was preserved and used by our laboratory, and cultivated with PDA.
[0057] 3.2 Botrytis infestation of tomato fruit
[0058] Wild type Botrytis cinerea B05.10 (WT) was cultured with PDA medium for 10-12 days to produce spores.
[0059] The change of pathogenicity of mutant strains was evaluated by fruit inoculation method. Botrytis cinerea spores were scraped off, eluted with Triton X-100 sterile aqueous solution and fil...
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