Map-based cloning reveals the complex organization of the BnRf locus and leads to the identification of BnRf b, a male sterility gene, in Brassica napus
Map-based cloning reveals the complex organization of the BnRf locus and leads to the identification of BnRf b, a male sterility gene, in Brassica napus
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DOI：
10.1007/s00122-015-2608-8
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发表时间：
2016-01-01
影响因子：
5.4
通讯作者：
Yang, Guangsheng
中科院分区：
文献类型：
--
作者：
Deng, Zonghan;Li, Xi;Yang, Guangsheng
甘蓝型油菜核雄性不育系9012 A的雄性不育性被认为是由BnMs 3/Bnms 3和包含3个等位基因的多等位基因BnRf基因所决定的。我们先前将BnRf定位于B上的13.8 kb DNA片段。油菜A7染色体。在本研究中，我们分离了分别覆盖恢复等位基因BnRf（a）和雄性不育等位基因BnRf（B）的细菌人工染色体克隆，并揭示了BnRf（a）和BnRf（B）的候选区域相对于保持等位基因BnRf（c）显示出复杂的结构变异。通过分析重组事件和新开发的标记，我们将BnRf（a）界定为一个35.9kb的DNA片段，该片段包含7个预测的开放阅读框（ORF）。然而，从雄性不育系和恢复系的ORF G14到野生型拟南芥植物的遗传转化导致了一个稳定的雄性不育表型匹配9012 A衍生的不育系（RG 206 A），而且，G14引起的雄性不育可以完全恢复恢复基因BnMs 3。这些事实表明BnRf（B）对应于G14，而BnRf（a）可能与另一个侧翼ORF相关。G14编码一种核定位的嵌合蛋白，命名为BnaA7.mtHSP70-1-like。G14基因在拟南芥中的异位表达可以负调控绒毡层退化的相关基因，延缓绒毡层细胞的程序性死亡，导致四分体的发育停滞。我们的工作不仅对不育控制的遗传模式提出了新的见解，而且为剖析9012 A雄性不育和恢复的分子基础奠定了坚实的基础。
The male sterility in an extensively used genic male sterility (GMS) line (9012A) in Brassica napus was regarded to be conferred by BnMs3/Bnms3 and the multiallelic BnRf locus including three alleles. We previously mapped BnRf to a 13.8 kb DNA fragment on the B. napus chromosome A7. In the present study, we isolated bacterial artificial chromosome clones individually covering the restorer allele BnRf (a) and the male-sterile allele BnRf (b) , and revealed that the candidate regions of BnRf (a) and BnRf (b) show complex structural variations relative to the maintainer allele BnRf (c). By analyzing the recombination events and the newly developed markers, we delimited BnRf (a) to a 35.9 kb DNA fragment that contained seven predicted open-reading frames (ORFs). However, genetic transformation of the ORF G14 from both the male-sterile and restorer lines into wild-type Arabidopsis plants led to a stable male-sterile phenotype matching a 9012A-derived GMS line (RG206A); moreover, the male sterility caused by G14 could be fully recovered by the restorer gene BnMs3. These facts indicate that BnRf (b) corresponds to G14 while BnRf (a) likely associates with another flanking ORF. G14 encodes a nucleus-localized chimeric protein designated as BnaA7.mtHSP70-1-like. Ectopic expression of G14 in Arabidopsis negatively regulates some vital genes responsible for tapetum degeneration, and delayed programmed cell death of tapetum and led to the developmental arrest of tetrads. Our work not only presents new insights on the hereditary model of sterility control but also lays a solid foundation for dissecting the molecular basis underlying male sterility and restoration in 9012A.