Resistance gene analog(RGA) screening of mapped disease-resistant genes not only helps to clone these genes but also helps to develop efficient molecular markers for resistance breeding. The present study focused on t...Resistance gene analog(RGA) screening of mapped disease-resistant genes not only helps to clone these genes but also helps to develop efficient molecular markers for resistance breeding. The present study focused on the PmU region located on chromosome 7 Au L of Triticum urartu, and recently, a nucleotide binding site(NBS)-encoding gene, Pm60, was cloned from the same chromosome arm. In this research, NBS, protein kinase(PK), and ATP-binding cassette(ABC), the three disease resistance-related gene families, were analyzed within PmU region by using informatics tools, and an expression experiment was conducted to verify their functions in vivo. Comparative genomic analysis revealed that 126 RGAs were included on chromosome 7 Au L, and 30 of the RGAs as well as Pm60 were found in the Pm U region. Transcriptome database analysis of T. urartu revealed 14 PmU-RGAs with expression data, and three PmU-NBSs exhibited significant changes in expression after inoculation with Blumeria graminis f. sp. tritici(Bgt); TRIUR314879 was up-regulated, while TRIUR300450 and TRIUR306270 were down-regulated. Cluster analysis showed that these three PmU-NBSs were clustered far from the cloned wheat resistance genes. Then, qRT-PCR was performed to investigate the expression of 14 PmU-RGAs and Pm60 after inoculation with Bgt race E09; the results showed that Pm60 was specifically expressed in UR206 which carrying PmU, but not in susceptible UR203; while TRIUR314879 was significantly up-regulated and TRIUR300450 was downregulated in UR206 after inoculation. These results indicated that PmU is Pm60, and TRIUR314879 and TRIUR300450 may also be involved in the defense against Bgt.展开更多
Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-...Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine protein kinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified from three wild rice species in Yunnan Province, China. The DIN A fragments from amplification have been cloned into the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. , and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Oryza officinalis Wall, and TR19 from Oryza rufipogon Griff, belong to the same class and homology of their sequences are 100%. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. 5 classes STK disease resistance gene analogues were also obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. By comparison analysis of amino acid sequences. we found that the obtained disease resistance gene analogues have very low identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative disease resistance genes that have not been isolated so far.展开更多
基金supported by the National Natural Science Foundation of China(31601307)the Key Scientific and Technological Innovation Platform of the Main Crop Germplasm Innovation and Molecular Breeding in Shanxi Province,China(201605D151002)the Youth Foundation of Institute of Crop Science,Shanxi Academy of Agricultural Sciences(ZZQ1701)
文摘Resistance gene analog(RGA) screening of mapped disease-resistant genes not only helps to clone these genes but also helps to develop efficient molecular markers for resistance breeding. The present study focused on the PmU region located on chromosome 7 Au L of Triticum urartu, and recently, a nucleotide binding site(NBS)-encoding gene, Pm60, was cloned from the same chromosome arm. In this research, NBS, protein kinase(PK), and ATP-binding cassette(ABC), the three disease resistance-related gene families, were analyzed within PmU region by using informatics tools, and an expression experiment was conducted to verify their functions in vivo. Comparative genomic analysis revealed that 126 RGAs were included on chromosome 7 Au L, and 30 of the RGAs as well as Pm60 were found in the Pm U region. Transcriptome database analysis of T. urartu revealed 14 PmU-RGAs with expression data, and three PmU-NBSs exhibited significant changes in expression after inoculation with Blumeria graminis f. sp. tritici(Bgt); TRIUR314879 was up-regulated, while TRIUR300450 and TRIUR306270 were down-regulated. Cluster analysis showed that these three PmU-NBSs were clustered far from the cloned wheat resistance genes. Then, qRT-PCR was performed to investigate the expression of 14 PmU-RGAs and Pm60 after inoculation with Bgt race E09; the results showed that Pm60 was specifically expressed in UR206 which carrying PmU, but not in susceptible UR203; while TRIUR314879 was significantly up-regulated and TRIUR300450 was downregulated in UR206 after inoculation. These results indicated that PmU is Pm60, and TRIUR314879 and TRIUR300450 may also be involved in the defense against Bgt.
文摘Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine protein kinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified from three wild rice species in Yunnan Province, China. The DIN A fragments from amplification have been cloned into the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. , and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Oryza officinalis Wall, and TR19 from Oryza rufipogon Griff, belong to the same class and homology of their sequences are 100%. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. 5 classes STK disease resistance gene analogues were also obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. By comparison analysis of amino acid sequences. we found that the obtained disease resistance gene analogues have very low identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative disease resistance genes that have not been isolated so far.
