Mutation And Differential Display Of Herbicidal-related Genes Of Botrytis Cinerea

It was reported that mycotoxins from Botrytis cinerea can be developed as microbial herbicides due to their high herbicidal bioactivity. However, the toxigenic heredity mechanism oiBotrytis cinerea is not clear at present. The fungi Botrytis cinerea is a known pathogen of tomato gray mould. In this thesis herbicidal substance and the genes of Botrytis cinerea that regulated toxin producing were studied by isolate BC4 and its mutant isolates. The main results are as follows:(1) Treated by ultraviolet ray and chemical mutagen, 50 isolates were obtained from isolate BC4's conidial suspension. Screening by biometrics and HPLC (highly effective liquid phase chromatography), three isolates (B1, B2, B3) with higher herbicidal activity and two isolates (B5, B23) with little herbicidal activity were selected out.(2) HPLC conditions were studied, and the results of HPLC analysis showed that the herbicidal content in isolates Bl, B2, and B3 were much higher than that in the wild isolate. Their biggest absorption peak value is at least five times higher than that in the wild isolate. But the quantity of each component in strain B5 and B23 is obviously dropped compared with wild isolate. At the same time we found their herbicidal activity did not change after they were cultured 8 generations. Optimized HPLC conditions were determined, and four fractions from the metabolite of the mutant isolates were obtained. Among them, the fraction I and IV had strong herbicidal activity to Digitaria sanguinalis and Amaranthus retroflexus.(3) In order to find out the molecular mechanism of herbicidal metabolite producing in Botrytis cinerea, mRNA DDRT-PCR (differential display reverse transcription PCR) was used to analysis the expressed genes of isolate BC4 and its mutant isolates. Differential displayed cDNA fragments that related to herbicidal activity were separated;Positive clones were selected out according to reverse northern dot blotting and some of them were sequenced.An optimum mRNA differential display protocol for Botrytis cinerea wasestablished. Combining 3 anchored Primers and 26 arbitrary primers, differential display pattern of the total RNA from the mutant isolates and the wild isolate was shown. Through comparing of strong herbicidal isolates and the weakly ones, 81 different displayed fragments were separated. They can be reamplified and 30 of them were confirmed as positive clones by reverse northern dot blotting and then 5 fragments were sequenced. Sequence analysis showed that, some fragments had high homology to calcium/calmodulm dependent protein kinase.(4) The unknown 5'sequence of fragment 49 was obtained by rapid amplification of cDNA ends. Special primers were designed by the 49's primary sequence. By 5' SMART? RACE cDNA amplification, a 1648bp fragment was obtained. And then the 694bp fragment was extended to 2055bp by compared with the former sequence. Sequence analysis showed that it has high similarity to calcium/calmodulin dependent protein kinase, calcium/calmodulin dependent protein kinase C, TRAPP complex subunit Bet5 and so on.