Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf4 bHLH transcription factor
Fertility restoration of maize CMS-C altered by a single amino acid substitution within the Rf4 bHLH transcription factor
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DOI：
10.1111/tpj.14521
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发表时间：
2019-10-25
期刊：
影响因子：
7.2
通讯作者：
Kumpatla, Siva P.
中科院分区：
文献类型：
--
作者：
Jaqueth, Jennifer S.;Hou, Zhenglin;Kumpatla, Siva P.
C型细胞质雄性不育（CMS-C）是玉米杂交种制种中最常用的细胞质雄性不育类型。育性恢复基因4（Rf 4）是CMS-C的主效育性恢复基因，位于8 S染色体上。为了定位克隆Rf 4，对来自非恢复系和恢复系之间杂交的大F3群体（n = 5104）筛选重组体，然后对雄穗育性进行表型分析，得到12 kb的最终基于图位的克隆间隔。在这12 kb的间隔内，Rf 4的唯一可能的候选者是GRMZM 2G 021276，一种具有雄穗特异性表达的碱性螺旋-环-螺旋（bHLH）转录因子。Rf 4基因产物含有核定位信号，并且可能不直接与线粒体相互作用。Rf 4的序列分析显示，恢复和非恢复自交系之间的4个编码氨基酸的取代，但只有一个取代，F187 Y，是在高度保守的bHLH结构域。通过使用成簇的规则间隔短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）同源定向修复（HDR）来测试Rf 4恢复被单个氨基酸改变的假设，以创建针对F187 Y取代而变化的等基因系。在对雄穗育性进行表型分析的这些CRISPR-Cas9编辑的植物（n = 780）的群体中，含有F187的植物是完全能育的，表明育性恢复，而含有Y187的植物是不育的，表明缺乏育性恢复。结构建模表明，该氨基酸残基187位于四螺旋束核心内，这是稳定二聚体构象和影响相互作用伴侣选择的关键区域。
Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.