Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis
Characterization of gamma irradiation-induced deletion mutations at a selectable locus in Arabidopsis
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DOI：
10.1016/s0027-5107(98)00009-8
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发表时间：
1998-06-05
影响因子：
2.3
通讯作者：
Milner, JJ
中科院分区：
文献类型：
--
作者：
Cecchini, E;Mulligan, BJ;Milner, JJ
以20-120 krad的剂量范围对含有负选择性自杀标记（作为转基因引入的35 S：：tms 2构建体）的转基因拟南芥种子进行γ-辐照。批次的M2种子，从M1植物照射的剂量为40，45和60 krad，筛选发芽的条件下，选择性地抑制生长的植物表达的tms 2基因产物的培养基中含有NAM。分离出9个候选的转基因缺失突变体。此类突变的频率（0.0125 - 0.025%）不随辐照剂量或M1合并液大小而显著变化。使用与转基因的各个区域互补的探针，在Southern印迹中杂交来自突变体和亲本的DNA。所有9个突变体对于tms 2编码序列和35 S启动子都是无效的。9个突变体中的6个对于9 kbp的整个转基因构建体是无效的。来自一个突变体的DNA含有一个T-DNA边界，并给出了与至少5 kbp的缺失一致的杂交模式。剩余的两个突变株系给出了相同的杂交模式，与转基因内5.6-kbp的内部缺失一致。从Southern印迹和谱系的基础上，九个株系代表七个或八个独立突变的后代。我们已经建立了能够产生至少5 kbp的缺失突变的条件，但显然不会引入小的缺失或重排。这样的缺失突变理想地适合于通过消减杂交进行克隆，并且还应该容易通过RFLP分析检测，从而促进基于图位的克隆程序。(C)1998 Elsevier Science B. V.保留所有权利。
Seeds of transgenic Arabidopsis, containing a negatively selectable suicide marker, a 35S :: tms2 construct introduced as a transgene, were gamma-irradiated at a range of doses from 20-120 krad. Batches of M2 seeds, from M1 plants irradiated at doses of 40, 45 and 60 krad, were screened by germinating them on medium containing NAM under conditions that selectively inhibited growth of plants expressing the tms2 gene product. Nine candidate loss-of-transgene mutants were isolated. The frequency of such mutations (0.0125 to 0.025%) did not vary significantly with irradiation dose or M1 pool size. DNA from the mutants and the parent was hybridized in Southern blots, using probes complementary to various regions of the transgene, All nine mutants were null for both the tms2 coding sequence and the 35S promoter. Six of the nine mutants were null for the entire transgene construct of 9 kbp. DNA from one mutant contained one of the T-DNA borders and gave a hybridization pattern consistent with a deletion at least 5 kbp. The two remaining mutant lines gave identical patterns of hybridization, consistent with a 5.6-kbp internal deletion within the transgene, From the Southern blots, and on the basis of lineage, the nine lines represent the progeny of either seven or eight independent mutations. We have established conditions capable of producing deletion mutations of at least 5 kbp, but without apparently introducing small deletions or rearrangements. Such deletion mutations are ideally suited for cloning by subtractive hybridization, and should also be readily detectable by RFLP analysis, facilitating map-based cloning procedures. (C) 1998 Elsevier Science B.V. All rights reserved.