利用BSA-Seq方法定位一个水稻早衰相关基因OsBRCA1

Mapping of Early Senescence-related OsBRCA1 in Rice by BSA-seq Technique

【目的】水稻早衰突变体是研究水稻衰老机制的良好载体。定位和克隆水稻早衰相关基因,有助于理解水稻早衰的遗传规律和分子机制,为相关基因的作用机制研究奠定基础。【方法】用EMS化学诱变处理粳稻云引,获得一个稳定遗传的早衰突变体w14,与日本晴杂交构建F;分离群体,并在群体中各选择100个隐性单株和显性单株的DNA等量混合,利用BSA-seq分析两个DNA池之间的差异位点,定位水稻早衰相关基因。【结果】突变体w14的早衰表型受单隐性基因控制,对两个DNA池测序结果进行单基因定位和突变基因的鉴定,发现单基因主峰位于Chr3,候选区间为Chr3:27.5~29.5 Mb,进一步在目标区域内找到2个符合条件的候选因果变异。【结论】候选基因LOC_Os03g49210突变位点是由野生型的C突变为T,且该突变位于候选基因的第2外显子上,属于错义突变,造成了该基因编码的第20个氨基酸由T(苏氨酸)变成I(异亮氨酸),从而可能导致了基因功能的改变,因此,将该基因定为本研究的候选基因,因与人类BRCA1同源,命名为OsBRCA1。

【Objective】Molecular mechanism of rice senescence was studied by locating and cloning the specific gene from a mutant artificially induced with early senescence.【Methods】Japonica rice Yunyin was treated with EMS to artificially induce a genetically stable early senescence w14.The mutant was crossed with Nipponbare to construct an isolated F;population.Subsequently,equal parts of the DNA of 100 recessive and dominant plants in the population were mixed.The differential loci between these two DNA pools were analyzed by BSA-seq to locate the target early senescence-related gene.【Results】The early senescence phenotype of w14 was found to be controlled by a single recessive gene.The mutagenic gene mapped and identified by the specific sequence showed the main peak located in Chr 3 and the candidate interval Chr 3:27.5–29.5 Mb.In the region,two qualified candidate causal variants were found.【Conclusion】The mutation site of the candidate gene Loc_Os03g49210 located in the 2^(nd) exon was determined to have changed in the wild-type from C to T.It was a non-synonymous mutation that resulted in the 20;amino acid encoded by the gene changed from T(threonine)to I(isoleucine)and led to the functional alternation.Being homologous with human BRCA1,the target gene was named OsBRCA1.