Expression analysis of Fusarium. genes involved in mycotoxin analysis. Erik Lysøe Fusarium, genes, and toxins 2008

Transcription

1 Expression analysis of Fusarium genes involved in mycotoxin analysis - experiments explained Erik Lysøe Fusarium, genes, and toxins 2008

2 Problem toxins in food and feed Approx. 400 known mycotoxins Fusarium toxins in cereals: Trichothecenes: - Deoxynivalenol (DON) - Nivalenol (NIV) - T-2 toxin - HT-2 toxin Zearalenone (ZON) Fumonisin Others: Aflatoxin - Aspergillus - Cereals (maize, sorghum, pearl millet, rice, wheat), oilseeds (peanut, soybean, sunflower, cotton), spices (chile peppers, black pepper, coriander, turmeric, ginger), and tree nuts (almond, pistachio, walnut, coconut, brazil nut). Ergotoxin - Claviceps purpurea - Cereals, rye (most common), triticale, wheat and barley. It affects oats only rarely. Ochratoxin A - Aspergillus, Penicillium - Cereals, coffee, beer, dried fruit, wine, cocoa and nuts. Patulin - Penicillium, Aspergillus and Byssochlamys - Apples

3 Genes involved in mycotoxin synthesis Trichothecenes Zearalenone Enniatin Haese, A., Schubert M., Herrmann M. and Zocher R. (1993). Molecular characterization of the enniatin synthetase t gene encoding a multifunctional l enzyme catalyzing N-methyldepsipeptide tid formation in Fusarium scirpi. Molecular Microbiology, 7, Fusarin C Song, Z.S., Cox R.J., Lazarus C.M. and Simpson T.J. (2004). Fusarin C biosynthesis in Fusarium moniliforme and Fusarium venenatum. Chembiochem, 5, Aurofusarin

4 Trichothecenes The Fusarium toxins most strongly assosiated with chronic and fatal toxicoses in humans and animals. Inhibition of ribosomal protein synthesis Two known gene clusters Two single genes

5 Tri5 Trichodiene synthase

6 T i h th Trichothecene genes hi highly hl expressed d in i planta l t Trichothecene genes Wheat Barley CMC Myro Conidia Asco CM,ND,CD Perithecia Conidia Asco CM,ND,CD Perithecia Aurofusarin genes Wheat Barley CMC Myro

7 Real-time experiments Trichothecene primers ELTri5a ELTri6a (regulator) Aurofusarin primers ELAurR1a (regulator) Housekeeping genes UBC EF1α Btub Samples Wheat control, 2d, 4d and 6d after inoculation with F. graminearum 3 replicate experiments

8 Real-time experiments Trizol RNA extraction Quality, gel Concentration, spectrophotometer or fluorometer DNAse treatment Clean-up of total RNA RNEasy column Quality and concentration, Bioanalyzer cdna synthesis Real-time PCR using SybrGreen Calculating primer efficiency Analyse results

9 Real-time experiments Pipetting of cdna, primers, water and SybrGreen Analyse results with Pfaffl method 1. Make standard d curve to calculate l primer efficiency i (E value) 2. Combine housekeeping genes into one value using BestKeeper 3. Analyse fold changes using REST program and Erik s graph maker

10

11 Using Excel

12

13 1. BestKeeper with example: Pfaffl, M.W., Tichopad, A., Prgomet, C., Neuvians, T.P., Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using gp pair-wise correlations. Biotechnol. Lett. 26, REST with example: Pfaffl, M.W., Tichopad, A., Prgomet, C., Neuvians, T.P., Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using gp pair-wise correlations. Biotechnol. Lett. 26, Erik s real-time graph maker with example If time, also learn the 2 ΔΔC T(Livak) method, example found in the lecture Introduction to real-time PCR All real-time files available on:

14 BestKeeper

15 REST

16 Expression pattern in a gene cluster using REST - normalizing against one gene (FG12056, WT, 7d)

17 Erik s graph maker Single CT values for all replicates Single CT values for all replicates Advantages: Standard deviation for the fold changes differences on the replicate runs Standard/log plot with error bars Minimum, maximum and median values Whiskers plot (Box plot)