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
Barley yellow dwarf virus (BYDV) is one of the most serious wheat diseases in China. So far no resistance has been described in common wheat. A certain level of BYDV resistance was found in thirteen Triticeae species....Barley yellow dwarf virus (BYDV) is one of the most serious wheat diseases in China. So far no resistance has been described in common wheat. A certain level of BYDV resistance was found in thirteen Triticeae species. Thinopyrum intermedium, two octoploids derived from TH. intermedium/wheat, Zhong 4 awnless and TAF46, and one disomic addition line, L1 derived from TAF46, showed good resistance to BYDV by enzyme linked immunosorbent assay (ELISA). Two wheat/TA. intermedium translocation lines, CPI 119880 and CPI 119899, showing good BYDV resistance were developed from L1 by using both CSph mutant and tissue culture. It is found that their BYDV resistance was controlled by a single dominant gene. Two cDNA probes pEleAcc3 and pPJN8 (E1-T1) were screened for detecting Th. intermedium DNA in wheat background. A specific band for the DNA of Th. intermedium and its derivatives was found in Southern hybridization. It is also possible to determine the size of the alien segment by comparing the relative density of the specific band. Therefore, this can be used as a marker to identify the BYDV resistance in wheat breeding program.展开更多
Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for...Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein(CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines(i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies(MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigencoated-plate enzyme-linked immunosorbent assay(ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840(w/v,g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120(w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.展开更多
In fission yeast (Schizosaccharomyces pombe), pac1 gene was cloned with 99.3% nucleo- tide sequence similarity with published pac1 in GenBank. In pET-5α expression system, the expres- sion product of cloned pac1 in E...In fission yeast (Schizosaccharomyces pombe), pac1 gene was cloned with 99.3% nucleo- tide sequence similarity with published pac1 in GenBank. In pET-5α expression system, the expres- sion product of cloned pac1 in E. coli showed activity to degrade the double-strand RNA. Harboring the binary vector pBI121, which contains pac1 gene, Agrobacterium tumefaciens strain LBA4404 was used to transform the wheat immature embryos pre- cultured 7―10 d. After preregeneration, regeneration and selection culture stage, totally 41 G418 resistant plants were obtained, in which 25 lines were proved to integrate with transgene and express transgene normally by PCR, Dot blot, RT-PCR and ELISA de- tection. Antivirus test carried out on 25 positive lines with high dose of Barley yellow dwarf virus-GPV re- vealed that 12 lines had resistance to BVDV-GPV in low level, another 12 lines had resistance to BVDV- GPV in middle level, and 1 line showed resistance to BVDV-GPV in high level. However, both low and middle level of resistance plants showed no symp- toms when infected by viruses at low dose, which suggested the dose-dependent effect of the resis- tance mediated by pac1 to BYDV-GPV.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.
基金This research is supported by the Australian Centre for International Agricultural Research(Projects 8379 and 8813)by the National Science and Technology Committee of China on China's side.
文摘Barley yellow dwarf virus (BYDV) is one of the most serious wheat diseases in China. So far no resistance has been described in common wheat. A certain level of BYDV resistance was found in thirteen Triticeae species. Thinopyrum intermedium, two octoploids derived from TH. intermedium/wheat, Zhong 4 awnless and TAF46, and one disomic addition line, L1 derived from TAF46, showed good resistance to BYDV by enzyme linked immunosorbent assay (ELISA). Two wheat/TA. intermedium translocation lines, CPI 119880 and CPI 119899, showing good BYDV resistance were developed from L1 by using both CSph mutant and tissue culture. It is found that their BYDV resistance was controlled by a single dominant gene. Two cDNA probes pEleAcc3 and pPJN8 (E1-T1) were screened for detecting Th. intermedium DNA in wheat background. A specific band for the DNA of Th. intermedium and its derivatives was found in Southern hybridization. It is also possible to determine the size of the alien segment by comparing the relative density of the specific band. Therefore, this can be used as a marker to identify the BYDV resistance in wheat breeding program.
基金supported by Public Science and Technology Research Funds Projects of Agriculture (201303021)the National Basic Research Program (973) of China (No.2014CB138400)
文摘Wheat dwarf disease caused by wheat dwarf virus(WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein(CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines(i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies(MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigencoated-plate enzyme-linked immunosorbent assay(ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840(w/v,g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120(w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.
文摘In fission yeast (Schizosaccharomyces pombe), pac1 gene was cloned with 99.3% nucleo- tide sequence similarity with published pac1 in GenBank. In pET-5α expression system, the expres- sion product of cloned pac1 in E. coli showed activity to degrade the double-strand RNA. Harboring the binary vector pBI121, which contains pac1 gene, Agrobacterium tumefaciens strain LBA4404 was used to transform the wheat immature embryos pre- cultured 7―10 d. After preregeneration, regeneration and selection culture stage, totally 41 G418 resistant plants were obtained, in which 25 lines were proved to integrate with transgene and express transgene normally by PCR, Dot blot, RT-PCR and ELISA de- tection. Antivirus test carried out on 25 positive lines with high dose of Barley yellow dwarf virus-GPV re- vealed that 12 lines had resistance to BVDV-GPV in low level, another 12 lines had resistance to BVDV- GPV in middle level, and 1 line showed resistance to BVDV-GPV in high level. However, both low and middle level of resistance plants showed no symp- toms when infected by viruses at low dose, which suggested the dose-dependent effect of the resis- tance mediated by pac1 to BYDV-GPV.
