Department of Biological Sciences, Seoul National University, Seoul 08826, Korea.
Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
Mol Cells. 2020 May 31;43(5):448-458. doi: 10.14348/molcells.2020.2290.
T-DNA insertional mutations in genes have conferred huge benefits to the research community, greatly facilitating gene function analyses. However, the insertion process can cause chromosomal rearrangements. Here, we show an example of a likely rearrangement following T-DNA insertion in the () gene locus on chromosome 5, so that the phenotype was not relevant to the gene of interest, . ASF1 is a histone H3/H4 chaperone involved in chromatin remodeling in the sporophyte and during reproduction. Plants that were homozygous for mutant alleles or were developmentally normal. However, following self-fertilization of double heterozygotes ( hereafter ), defects were visible in both male and female gametes. Half of the and ovules displayed arrested embryo sacs with functional megaspore identity. Similarly, half of the and pollen grains showed centromere defects, resulting in pollen abortion at the bi-cellular stage of the male gametophyte. However, inheritance of the mutant allele in a given gamete did not solely determine the abortion phenotype. Introducing functional ASF1B failed to rescue the and mediated abortion, suggesting that heterozygosity in the gene causes gametophytic defects, rather than the loss of . The presence of reproductive defects in heterozygous mutants but not in homozygotes, and the characteristic all-or-nothing pollen viability within tetrads, were both indicative of commonly-observed T-DNA-mediated translocation activity for this allele. Our observations reinforce the importance of complementation tests in assigning gene function using reverse genetics.
T-DNA 插入突变在基因中赋予了研究界巨大的好处，极大地促进了基因功能分析。然而，插入过程会导致染色体重排。在这里，我们展示了一个在 5 号染色体上的 ()基因座中 T-DNA 插入后可能发生的重排的例子，因此表型与感兴趣的基因（）无关。ASF1 是一种组蛋白 H3/H4 伴侣，参与孢子体和繁殖过程中的染色质重塑。纯合突变等位基因 或 的植物在发育上是正常的。然而，在双杂合子（以下简称）自交后，雄性和雌性配子中都出现了缺陷。一半的 或 胚珠显示出具有功能性大孢子身份的胚胎囊停滞。同样，一半的 或 花粉粒显示着丝粒缺陷，导致雄性配子体的二细胞阶段花粉败育。然而，在给定的配子中突变等位基因的遗传并不单独决定流产表型。引入功能正常的 ASF1B 并不能挽救 或 介导的流产，这表明 基因的杂合性导致配子体缺陷，而不是 的丢失。杂合突变体中存在生殖缺陷，但纯合子中没有，四分体中花粉活力的全或无特征，都表明该等位基因存在常见的 T-DNA 介导的易位活性。我们的观察结果强调了在使用反向遗传学分配基因功能时进行互补测试的重要性。
