Hanley S, Edwards D, Stevenson D, Haines S, Hegarty M, Schuch W, Edwards K J
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, UK.
Plant J. 2000 Aug;23(4):557-66. doi: 10.1046/j.1365-313x.2000.00830.x.
We have used a universal adaptor amplification procedure to isolate random Mutator-tagged fragments from Mutator-active maize plants. Direct sequence characterization of 761 Mutator-tagged fragments indicated that a significant number were homologous to sequences within the public databases. The ability of Mutator-tagged fragments to detect homology was not related to the length of the sequence within the range 100-400 bp. However, fragments above this size did show an increased chance of detecting homology to either expressed sequence tags or genes. Characterization of the insertion sites of the Mutator elements suggested that while it does target transcribed regions, Mutator does not appear to have any site preference within the transcription unit. Hybridization of previously unidentified Mutator-tagged fragments to arrayed cDNA libraries confirmed that many of these also showed homology to transcribed regions of the genome. Examination of back-crossed progeny confirmed that all the insertions examined were germinal; however, in all but one case, selfing five individual Mutator-tagged lines failed to reveal an obvious phenotype. This study suggests that the random sequencing of Mutator-tagged fragments is capable of producing both a significant number of interesting transposon tagged genes and mutant plant lines, all of which could be extremely valuable in future gene discovery and functional genomics programmes.
我们采用了一种通用衔接子扩增程序，从具有Mutator活性的玉米植株中分离随机的Mutator标签片段。对761个Mutator标签片段进行直接序列特征分析表明，相当数量的片段与公共数据库中的序列同源。Mutator标签片段检测同源性的能力与100 - 400 bp范围内的序列长度无关。然而，大于这个大小的片段确实显示出与表达序列标签或基因检测到同源性的几率增加。对Mutator元件插入位点的特征分析表明，虽然它确实靶向转录区域，但Mutator在转录单元内似乎没有任何位点偏好。将先前未鉴定的Mutator标签片段与阵列cDNA文库杂交证实，其中许多片段也与基因组的转录区域显示出同源性。对回交后代的检查证实，所有检测的插入都是生殖性的；然而，除了一个案例外，对五个单独的Mutator标签系进行自交未能揭示明显的表型。这项研究表明，对Mutator标签片段进行随机测序能够产生大量有趣的转座子标签基因和突变植株系，所有这些在未来的基因发现和功能基因组学计划中都可能极具价值。
