Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncate...Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication- associated （RepA） gene （WDVRepA215） and the WDV RepA gene with a mutated retinoblastoma-related protein （RBR） interaction domain （WDVRepA215RBRre^t） were cloned into the plPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers （Psammotettix alienus） fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBR^mut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBR^mut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.展开更多
Limen is a small cysteine-rich peptide that was originally isolated from lima beans,and its activity against Micromycetes was described.Recombinant expression of limen in both a prokaryotic and plant system was perfor...Limen is a small cysteine-rich peptide that was originally isolated from lima beans,and its activity against Micromycetes was described.Recombinant expression of limen in both a prokaryotic and plant system was performed to demonstrate a wide selection of its activities with potential for further use.At first limen was expressed in E.coli cells and after purification steps,its antimicrobial activity was tested on pathogenic fungi.This showed that limen can significantly eliminate the growth of several pathogenic yeasts such as Candida albicans,while its hemolytic activity was not recognizable.To confirm limen’s activity in vivo,transformation of the barley Hordeum vulgare L.was chosen.Transgenic barley was obtained using Agrobacterium-mediated transformation and the production of limen peptide in plant tissues was verified by molecular techniques.Young barley plants were repeatedly exposed to Fusarium oxysporum spores.Increased resistance to the most important pathogen was followed using chlorophyll measurements as indicators of fusariosis.Focusing on the food safety aspect of the use of GM plants,cytotoxicity was followed in extracts from transgenic stressed plants.No or a lower cytotoxic effect was found on human keratinocytes and human embryonic kidney cells than extracts from the non-transformed plants.展开更多
基金the Czech Ministry of Education, Youth and Sports funding programme LH12161the Czech Ministry of Agriculture funding programme MZE RO0417
文摘Wheat dwarf virus （WDV）, an important cereal pathogen, is closely related to Maize streak virus （MSV）, a model virus of the Mastrevirus genus. Based on its similarity to known MSV resistance strategies, a truncated part of the WDV replication- associated （RepA） gene （WDVRepA215） and the WDV RepA gene with a mutated retinoblastoma-related protein （RBR） interaction domain （WDVRepA215RBRre^t） were cloned into the plPKb002 expression vector and transformed into immature embryos of spring barley cv. Golden Promise plants through Agrobacterium-mediated transformation. A detailed study of T1-generation plants infected by leafhoppers （Psammotettix alienus） fed on infection sources of variable strength was performed over a 5-week period encompassing the initial stages of virus infection. A DNA WDV TaqMan qPCR assay normalized using the DNA puroindoline-b SYBR Green qPCR assay for samples on a per week basis revealed an approximately 2-week delay in WDVRepA215RBR^mut plants to WDVRepA215 plants before significant increases in the WDV viral levels occurred. Both WDVRepA215 and WDVRepA215RBR^mut plants showed similar levels of transgenic transcripts over the screened period; however, the transgenic plants also showed increased numbers of infected plants compared to the control plants.
文摘Limen is a small cysteine-rich peptide that was originally isolated from lima beans,and its activity against Micromycetes was described.Recombinant expression of limen in both a prokaryotic and plant system was performed to demonstrate a wide selection of its activities with potential for further use.At first limen was expressed in E.coli cells and after purification steps,its antimicrobial activity was tested on pathogenic fungi.This showed that limen can significantly eliminate the growth of several pathogenic yeasts such as Candida albicans,while its hemolytic activity was not recognizable.To confirm limen’s activity in vivo,transformation of the barley Hordeum vulgare L.was chosen.Transgenic barley was obtained using Agrobacterium-mediated transformation and the production of limen peptide in plant tissues was verified by molecular techniques.Young barley plants were repeatedly exposed to Fusarium oxysporum spores.Increased resistance to the most important pathogen was followed using chlorophyll measurements as indicators of fusariosis.Focusing on the food safety aspect of the use of GM plants,cytotoxicity was followed in extracts from transgenic stressed plants.No or a lower cytotoxic effect was found on human keratinocytes and human embryonic kidney cells than extracts from the non-transformed plants.
