[Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR].
[Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR].
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发表时间：
2002-05
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通讯作者：
Genji Qin;Pei-du Chen;Yaoguang Liu;Y. Fang;Dajun Liu
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作者：
Genji Qin;Pei-du Chen;Yaoguang Liu;Y. Fang;Dajun Liu
根据抗病基因的NBS结构域设计了一对简并引物，对小麦-簇毛麦易位系6VS/6AL的cDNA进行PCR扩增。通过测序分析获得了具有NBS特征的克隆（N7）。根据N7序列设计了两条特异性引物，用于筛选6VS/6AL的基因组TAC（transformation-competent artificial chromosome，TAC）文库。2 x 10（6）个克隆。将文库作为克隆池储存在22个96孔板中，每个孔含有约1000个TAC克隆。从所有2112个池制备TAC质粒。使用合并的PCR筛选程序，获得具有40 kb插入片段的阳性TAC克隆。以NBS片段为探针进行Southern杂交，证实阳性克隆。结果表明，合并PCR方法是筛选具有大量克隆的基因组文库的有效方法。
A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.