Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector
Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector
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DOI：
10.1046/j.1365-313x.2000.00827.x
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发表时间：
2000-09-01
期刊：
影响因子：
7.2
通讯作者：
Ogihara, Y
中科院分区：
文献类型：
--
作者：
Liu, YG;Nagaki, K;Ogihara, Y
普通小麦、小麦和小麦的三个大插入基因组DNA文库。在大肠杆菌和农杆菌中稳定地接受和维持大片段基因组DNA的TAC载体pYLTAC17上构建了中国春。该载体包含农杆菌介导的基因转移到草中所需的顺式序列。三个基因组文库的平均插入长度分别为46、65和120 kbp，覆盖了三个单倍体基因组。基因组文库以96孔格式冷冻培养保存，每孔含有约300-600个菌落（小型文库12个，中型文库4个，大型文库4个）。在每个文库中，大约80%的菌落都含有大约50 kbp的基因组DNA插入物。用聚合酶链反应（pooled PCR）技术鉴定含有目标基因的TAC克隆。一旦目标TAC克隆被分离出来，它们可以立即通过农杆菌系统转移到草基因组中。利用聚合酶链反应（pooled PCR）方法，成功筛选出A、B、D三个基因组对应的5个含硫蛋白I型基因（单拷贝）的克隆，以及一个STS标记（aWG464，单拷贝）和CAB（一个多基因家族）。本文所述构建的TAC文库可用于分离含有目标基因的基因组克隆，并进行基因组行走以进行定位克隆。
Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv. Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses. The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents. Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library). In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of > 50 kbp. TAC clones containing gene(s) of interest were identified by the pooled PCR technique. Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system. Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family). TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning.