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Mutation and Differential Display of Herbicidal-related Genes of Botrytis Cinerea

Author: MaJuan
Tutor: DongJinGao；ZhangJinLin
School: Agricultural University of Hebei
Course: Plant Pathology
Keywords: Botrytis cinerea Herbicidal activity mRNA differential display method rapid amplification of cDNA ends
CLC: S482.4
Type: Master's thesis
Year: 2006
Downloads: 68
Quote: 1
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Abstract


Toxins produced by Botrytis having herbicidal activity, and is expected to develop into a microbial herbicide. The test on Botrytis cinerea the BC4 strains and mutant strains obtained by its mutagenesis material difference compare its toxigenic herbicidal activity and herbicidal active substances contained in the regulation of Botrytis cinerea herbicidal active substance-related genes The research results are as follows: 1. Botrytis cinerea BC4 starting strain, 50 strains were obtained by UV mutagenesis and chemical mutagens conidia. Seed germination law and foliar treatment screening, get the toxins herbicidal activity than the wild-type strain significantly enhanced three mutant strains B1, B2, B3 and herbicidal activity almost lost two mutant strains B5 and B23. Fumble chromatographic conditions for HPLC analysis of the toxins produced by various strains, and found that the content of each component in the B1, B2, B3 and other mutant strains were significantly higher than the wild-type strain, the maximum absorption peak height at five times the wild-type strain above. Strains B5, B23 toxin content of each component with the wild-type strain significantly decreased both the maximum peak height values ??the BC4 only 39% and 34%. Genetic stability tests showed that the tested strains genetically stable, continuous culture 8th generation herbicidal activity did not change. Optimize the HPLC conditions were isolated and purified strong herbicidal activity of toxins, prepared by four components, wherein component I and IV activity has a strong addition to killing crabgrass and Amaranthus retroflexus. By mRNA differential display reverse transcription BC4 of wild-type strain and mutant strains differential display analysis. Different strains of Botrytis cinerea genes differentially expressed fragments were separated, and then amplified by trans Northern hybridization detection, screened positive fragments were cloned, sequenced and analyzed. The test strains of Botrytis cinerea mRNA differential display system using three anchor primers and 26 random primer combinations, show differences in the expression of different strains of Botrytis cinerea gene fragment. Comparison between the strong herbicidal activity of strains with weak or no herbicidal activity strains, a total of 81 different fragments. Differential fragments purified to the same conditions and corresponding primers for the second amplification, can be effectively amplified fragment. By the trans Northern blot, 30 screened positive fragments. 5 difference expressed gene fragments were cloned and sequenced, the length of 462bp, 694bp, 173bp, 256bp and 156bp cDNA sequences using the BLAST software sequence analysis, found fragments 49 and calcium / calmodulin-dependent protein kinase with high homology. 4 using rapid amplification of cDNA ends (rapid amplification of cDNA ends, RACE) technology for the pre-obtained gene fragment 49 and the 5 'end of the unknown sequence was amplified. According to the cDNA sequence of the fragment 49, specific primers were designed, 5'RACE reaction to give a 1648bp fragment. The original fragment sequences spliced ??so that it extended to 2055bp. With calcium / calmodulin-dependent protein kinase, TRAPP complex subunit Bet5 and other protein has high similarity.

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CLC: > Agricultural Sciences > Plant Protection > Pesticide ( chemical control ) > Various pesticides > Herbicides ( herbicide )
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