OsMTOPVIB Promotes Meiotic DNA Double-Strand Break Formation in Rice
OsMTOPVIB Promotes Meiotic DNA Double-Strand Break Formation in Rice
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OsMTOPVIB 促进水稻减数分裂 DNA 双链断裂形成
DOI：
10.1016/j.molp.2016.07.005
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发表时间：
2016
期刊：
影响因子：
27.5
通讯作者：
Zhukuan Cheng
中科院分区：
文献类型：
--
作者：
Zhihui Xue;Yafei Li;Lei Zhang;Wenqing Shi;Chao Zhang;Mengshi Feng;Fanfan Zhang;Ding Tang;Hengxiu Yu;Minghong Gu;Zhukuan Cheng
减数分裂重组是由Spo11蛋白催化的DNA双链断裂(DSB)的产生启动的(De Massy，2013)。两个关键的发现指出了Spo11作为发芽酵母中DSB形成的启动子的作用：(1)Spo11与rad50s突变体中断裂DNA分子的50端相连，其减数分裂在切割反应阶段被阻断(Keeney等人，1997年)；(2)Spo11通过催化亚基与古生菌DNA拓扑异构酶(TopVIA)同源，后者属于IIB型拓扑异构酶家族(Bergerat等人，1997)。拓扑异构酶VI是一种异构体，由两个TopVIA分子和两个TopVIB(TopVIB)分子组成。TopVIA含有一个保守的酪氨酸，参与了DNA断裂形成的酯交换反应。TopVIB包含一个ATP结合域(GHKL)和一个转导器域(Corbett等人，2007年；Granle等人，2008年)。转导子结构域参与将ATP结合和水解引起的构象变化从GHKL转移到A亚基(Bates等人，2011年)。最近，拟南芥MTOPVIB和小鼠TOPOVIBL等拓扑类VIB蛋白被鉴定出来。MTOPVIB和TOPOVIBL与古菌TopVIB有结构上的同源性，是通过与Spo11相互作用形成DSB所必需的(Robert等人，2016；Vrielynck等人，2016)。在本研究中，我们克隆了一个水稻基因，命名为OsMTOPVIB，因为它与拟南芥MTOPVIB同源。我们的结果表明OsMTOPVIB对于减数分裂DSB的形成是必需的。从水稻品种延稻8号中鉴定到一个完全不育的突变体。突变体表现出正常的营养生长，但开花后表现为不育。它的花粉粒缩小，不能存活(补充图1)。当用野生型花粉给突变体的花授粉时，突变体没有结实任何种子，这表明突变体的雌配子也受到了损害。杂合子植株的可育株与不育株之比为3：1(可育株117株，不育株43株)，表明其表型来源于一次隐性突变(c2=0.30；P>；0.05)。通过基于图谱的克隆，目标基因被锚定在水稻6号染色体上39kb的区域(补充图2)。仅在候选基因(LOC_Os06g49450)中发现突变。通过基于图谱的克隆也分离到另外两个等位基因(补充图3B)。为了验证LOC_Os06g49450突变是否与突变体的表型有关，我们进行了基因特异性RNA干扰(RNAi)实验，发现大多数独立的转基因株系表现不育(95%，n=60)。
Meiotic recombination is initiated by the generation of DNA double-strand breaks (DSBs), which are catalyzed by the Spo11 protein (de Massy, 2013). Two key findings point to the role of Spo11 as the initiator of DSB formation in budding yeast:(1) Spo11 is linked to the 50 termini of the broken DNA molecules in rad50s mutants, whose meiosis has been blocked at the stage of the cleavage reaction (Keeney et al., 1997); and (2) Spo11 shares homology with the archaeal DNA topoisomerase VIA catalytic subunit (TopVIA), which belongs to a family of type IIB topoisomerases (Bergerat et al., 1997). Topoisomerase VI functions as a heterotetramer, composed of two molecules of TopVIA and two molecules of topoisomerase B (TopVIB). TopVIA contains a conserved tyrosine involved in the transesterification reaction for DNA break formation. TopVIB contains an ATP-binding domain (GHKL) and a transducer domain (Corbett et al., 2007; Graille et al., 2008). The transducer domain is involved in transferring the conformational changes induced by ATP binding and hydrolysis from GHKL to the A subunits (Bates et al., 2011). Recently, TopoVIB-like proteins, including Arabidopsis MTOPVIB and mouse TOPOVIBL, were identified. Both MTOPVIB and TOPOVIBL share structural homology with archaeal TopVIB and are required for DSB formation through an interaction with Spo11 (Robert et al., 2016; Vrielynck et al., 2016). In this study, we cloned a gene in rice, named OsMTOPVIB for its homology with Arabidopsis MTOPVIB. Our results show that OsMTOPVIB is essential for meiotic DSB formation.A completely sterile mutant was identified from Yandao 8, a japonica rice variety. The mutant exhibited normal vegetative growth but presented as sterile after flowering. Its pollen grains were shrunken and inviable (Supplemental Figure 1). When the mutant flowers were pollinated with wild-type pollen grains, the mutant did not set any seeds, suggesting that female gametes were also impaired in the mutant. The ratio of fertile to sterile plants derived from self-fertilization of the heterozygous plants was 3: 1 (fertile, 117; sterile, 43), suggesting that the phenotype is derived from a single recessive mutation (c2= 0.30; P> 0.05). By map-based cloning, the target gene was anchored to a 39-kb region on rice chromosome 6 (Supplemental Figure 2). A mutation was found only within the candidate gene (LOC_Os06g49450). Two additional alleles were also isolated through map-based cloning (Supplemental Figure 3B). To verify whether the mutation of LOC_Os06g49450 is responsible for the phenotype of the mutant, we performed gene-specific RNA interference (RNAi) experiments and found that most independent transgenic lines exhibited sterility (95%, n= 60).