A endochitinase gene（Tch） from the fungus Trichoderma viride was introduced into broccoli（Brassica oleracea var. italica） by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained ...A endochitinase gene（Tch） from the fungus Trichoderma viride was introduced into broccoli（Brassica oleracea var. italica） by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was confirmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression profiling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endochitinase using a fluorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was confirmed to be inheritable. These findings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.展开更多
The Hypocrea lixii/Trichoderma harzianum species aggregate contains a group of taxa (H. lixii /T. harzianum, T. aggressivum, T. tomentosum, T. cerinum, T. velutinum, H. tawa) of which some (e.g. T. harzianum) are impo...The Hypocrea lixii/Trichoderma harzianum species aggregate contains a group of taxa (H. lixii /T. harzianum, T. aggressivum, T. tomentosum, T. cerinum, T. velutinum, H. tawa) of which some (e.g. T. harzianum) are important for biocontrol of plant pathogenic fungi in agriculture, whereas others are aggressive pathogens of Agaricus spp. and Pleurotus spp. in mushroom farms (T. aggressivum), or opportunistic pathogens of immunocompromised mammals including humans (T. harzianum). We characterized the evolutionary properties of three genomic regions in Hypocrea/Trichoderma: the internal transcribed spacer regions ITS1 and 2 of rDNA, the large intron of translation elongation factor 1-alpha (tef1a), and a portion of the large exon of the endochitinase 42 gene (ech42), selected the best model which describes the evolution of every fragment, tested the molecular clock hypothesis and made an estimation of the usability of the combined three fragments data matrix for the phylogenetic analysis of the genus as a whole as well as on the level of the holomorphic H. lixii/T. harzianum species clade and separate clonal lineages. To this end, we applied Bayesian phylogenetic inferences to 124 sequences of ITS1 and 2 and of the large tef1a intron, and to 64 ech42 gene sequences to resolve the evolution of H. lixii/T. harzianum with respect to the position of other taxa with closely related phenotypes. The resulting phylogram clearly identified T. aggressivum, T. velutinum, H. tawa, T. cerinum and T. tomentosum as phylogenetic species, and in addition identified three new unknown phylogenetic species as members of this clade. The clear distinction between T. tomentosum and T. cerinum was not recognized in all trees, but was supported by multivariate analysis of phenotype micro arrays. In contrast, H. lixii/T. harzianum did not form a single phylogenetic species in this study, as its monophyly was not supported in any analysis. Strains morphologically identified as H. lixii or T. harzianum formed either a proliferating megaclade (th展开更多
Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Tri...Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Trichoderma endochitinase gene ThEn-42 at initial bud stage. Eleven seedlings (corresponding to about 0.22 percent transformation) exhibited resistance to hygromycin. The DNA fragment unique to endochitinase (ThEn-42) was amplified by Arabidopsis leaf-PCR or genomic DNA PCR. Unfertile, dwarf and normal phenotypes appeared in the T1 generation. In addition, an enhanced resistance to S. sclerotiorum was observed. The mortality percentage (7.7% to 33.3%) in transgenic plants was significantly lower than in non-transgenic plants (86.7%) 10 days after inoculation with the pathogen.展开更多
基金partly supported by the 863 Program (2012AA100A03)973 Program (2012CB113900)Beijing Agricultural Industry Project, China (BLVT-03)
文摘A endochitinase gene（Tch） from the fungus Trichoderma viride was introduced into broccoli（Brassica oleracea var. italica） by Agrobacterium-mediated transformation. Sixty-eight putative transformants were obtained and the presence of the Tch gene was confirmed by both PCR and Southern blot analysis. RT-PCR analysis showed an accumulation of the transcript encoding the endochitinase protein in the transgenic plants. Using real-time quantitative PCR, the expression profiling of endochitinase gene was analyzed. Primary transformants and selfed progeny were examined for expression of the endochitinase using a fluorometric assay and for their resistance to the pathogenic fungi Botrytis cinerea and Rhizoctonia solani. The endochitinase activities in T0 in vitro plants, T0 mature plants and T1 mature plants were correlated with leaf lesions, and the transgenic line T618 had high endochitinse activities of 102.68, 114.53 and 120.27 nmol L–1 MU min–1 mg–1 protein in the three kinds of plants, respectively. The endochitinase activity showed a positive correlation with the resistance to the pathogens. Most transgenic T0 broccoli had increased resistance to the pathogens of B. cinerea and R. solani in leaf assays and this resistance was confirmed to be inheritable. These findings suggested that expression of the Tch gene from T. viride could enhance resistance to pathogenic fungi in Brassica species.
文摘The Hypocrea lixii/Trichoderma harzianum species aggregate contains a group of taxa (H. lixii /T. harzianum, T. aggressivum, T. tomentosum, T. cerinum, T. velutinum, H. tawa) of which some (e.g. T. harzianum) are important for biocontrol of plant pathogenic fungi in agriculture, whereas others are aggressive pathogens of Agaricus spp. and Pleurotus spp. in mushroom farms (T. aggressivum), or opportunistic pathogens of immunocompromised mammals including humans (T. harzianum). We characterized the evolutionary properties of three genomic regions in Hypocrea/Trichoderma: the internal transcribed spacer regions ITS1 and 2 of rDNA, the large intron of translation elongation factor 1-alpha (tef1a), and a portion of the large exon of the endochitinase 42 gene (ech42), selected the best model which describes the evolution of every fragment, tested the molecular clock hypothesis and made an estimation of the usability of the combined three fragments data matrix for the phylogenetic analysis of the genus as a whole as well as on the level of the holomorphic H. lixii/T. harzianum species clade and separate clonal lineages. To this end, we applied Bayesian phylogenetic inferences to 124 sequences of ITS1 and 2 and of the large tef1a intron, and to 64 ech42 gene sequences to resolve the evolution of H. lixii/T. harzianum with respect to the position of other taxa with closely related phenotypes. The resulting phylogram clearly identified T. aggressivum, T. velutinum, H. tawa, T. cerinum and T. tomentosum as phylogenetic species, and in addition identified three new unknown phylogenetic species as members of this clade. The clear distinction between T. tomentosum and T. cerinum was not recognized in all trees, but was supported by multivariate analysis of phenotype micro arrays. In contrast, H. lixii/T. harzianum did not form a single phylogenetic species in this study, as its monophyly was not supported in any analysis. Strains morphologically identified as H. lixii or T. harzianum formed either a proliferating megaclade (th
文摘Sclerotinia sclerotiorum is an important pathogen to many crops and is especially damaging to rape in China. As a model plant Arabidopsis thaliana (Col0) was transformed by spraying Agrobacterium tumefacience with Trichoderma endochitinase gene ThEn-42 at initial bud stage. Eleven seedlings (corresponding to about 0.22 percent transformation) exhibited resistance to hygromycin. The DNA fragment unique to endochitinase (ThEn-42) was amplified by Arabidopsis leaf-PCR or genomic DNA PCR. Unfertile, dwarf and normal phenotypes appeared in the T1 generation. In addition, an enhanced resistance to S. sclerotiorum was observed. The mortality percentage (7.7% to 33.3%) in transgenic plants was significantly lower than in non-transgenic plants (86.7%) 10 days after inoculation with the pathogen.
