用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆  

Screening for Resistance Gene Candidate from a Genomic TACLibrary of Triticum aestivum-Haynaldia villosa TranslocationLine 6VS/6AL by Pooled PCR

作　　者：秦跟基[1] 陈佩度[1] 刘耀光[2] 方玉达[1] 刘大钧[1] 

机构地区：[1]南京农业大学细胞遗传所,南京210095 [2]华南农业大学生命科学学院遗传工程研究室,广州510642

出　　处：《生物工程学报》2002年第3期313-317,共5页Chinese Journal of Biotechnology

基　　金：国家"8 6 3"高技术研究发展计划研究项目 (No Z 17 0 4 0 1);国家转基因植物研究与产业化专项 (No J0 0 A 0 0 2 )~~

摘　　要：用根据抗病基因保守区设计的一对简并性引物 ,从小麦 簇毛麦易位系 6VS 6ALcDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点 (Nucleotidebindingsite,NBS)结构特点的DNA片段克隆N7。从小麦 簇毛麦易位系6VS 6AL基因组TAC(Transformation competentartificialchromosome,TAC)文库的 2 2块 96孔板提取所有 2 112个克隆池(每个池含约 10 0 0个克隆 )的质粒 ,再根据N7的核苷酸序列设计一对特异引物 ,用克隆池PCR(pooledPCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针 ,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。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. 2×10 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.

关 键 词：克隆池PCR法 筛选 小麦-簇毛麦易位系 6VS/6AL 基因组TAC文库 抗病基因 基因克隆 

分 类 号：Q943.2[生物学—植物学] S512.1[农业科学—作物学]