Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from L...Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.展开更多
Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR ...Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.展开更多
基金the National Natural Science Foundation of China(Grant Nos.30370905 and 30571135)Natural Science Foundation of Beijing City(Grant No.5032009)
文摘Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.
基金supported by the National Natural Science Foundation of China (Grant No. 30760115)Transgenic Program (Grant No. 2008ZX08001-002)
文摘Based on the sequence of a resistance gene analog FZ14 derived from Zizania latifolia （Griseb.）, a pair of specific PCR primers FZ14P1/FZ14P2was designed to isolate candidate disease resistance gene. The pooled-PCR approach was adopted using the primer pair to screen a genomic transformation-competent artificial chromosome （TAC） library derived from Z. latifolia. A positive TAC clone （ZR1） was obtained and confirmed by sequence analysis. The results indicated that ZR1 consisted of conserved motifs similar to P-loop （kinase la）, kinase 2, kinase 3a and GLPL （Gly-Leu-Pro-Leu）, suggesting that it could be a portion of NBS-LRR type of resistance gene. Using Agrobacterium-mediated transformation of Nipponbare mature embryo, a total of 48 independent transgenic To plants were obtained. Among them, 36 plants were highly resistant to the virulent bacterial blight strain PXO71. The results indicate that ZR1 contains at least one functional bacterial blight resistance gene.
