ZmEREB180耐渍功能证实及调控机理研究

Waterlogging is one of the most important abiotic stress among the life cycle of maize. The previous projects had showed that the natural variation of ZmEREB180, a group VII ERF gene of maize, was significantly associated with waterlogging tolerance. Overexpression of ZmEREB180 in Arabidopsis and maize had verified that transgenic plants improved dramatically the survival ability under submerging and waterlogging condition, respectively. We have obtained the mutant of ZmEREB180, T2 generation seeds of overexpression and knockout of ZmEREB180 in maize. In this project, our objectives are i) to examine the botanic phenotype of mutant and transgenic lines under waterlogging condition to reveal the biological function of ZmEREB180; ii) to assay the specific location where ZmEREB180 is expression in the root by RNA in situ hybridization, analyze the nucleotide motif bound by ZmEREB180 through ChIP-seq and EMSA, reveal the interacting proteins by Co-IP and validate the interaction by the yeast-two hybrid system, compare the differentially-expressed genes by RNA-seq, combine all of the mentioned examinations results to illuminate the regulatory network of ZmEREB180; iii) to clarify origin and selection of prior alleles in the process of domestication and genetic improvement of maize by molecular evolution analysis. Our results will establish theoretical basis of illustrating the gene function, genetic effect and mechanism of waterlogging tolerance at maize seedling stage and provide prior gene sources for molecular breeding of waterlogging stress.

渍害是影响玉米生长发育重要的非生物逆境因子之一。项目组前期对玉米第七亚家族ERF基因研究发现，ZmEREB180的自然变异与玉米耐渍性显著关联。拟南芥和玉米过表达遗传转化证实，过表达该基因能够显著提高拟南芥和玉米在淹水胁迫下的存活能力。本项目拟在前期已获得ZmEREB180突变体及其在玉米中敲除遗传转化T2代种子的基础上，通过对突变体和遗传转化后代淹水胁迫下植物学表型分析，探明ZmEREB180生物学功能；利用原位杂交、RNA-seq、ChIP-seq、EMSA、Co-IP和酵母单双杂等方法，明确该基因特异表达的组织器官、特异结合的顺式元件及互作蛋白，阐明其参与的耐渍调控网络及其分子机制；通过分子进化分析，探明其在玉米驯化和遗传改良过程中优良等位基因的起源和选择模式。研究结果将为阐明玉米苗期耐渍性基因功能、遗传效应和作用机理提供重要的理论依据，为开展玉米耐渍分子育种提供优良等位基因资源。

渍害是影响玉米生长发育重要的非生物逆境因子之一。本项目通过候选基因关联分析鉴定到ZmEREB180正调控玉米耐渍性，利用酵母双杂交和LUC实验已证实ZmEREB180能够与ZmMPK1和ZmMPK3互作，通过转录组测序和酵母单杂交分析初步明确ZmEREB180直接结合到ZmLBD5和ZmRBOHD启动子上调控根系发育和活性氧平衡，利用酵母单杂交分析也已证实ZmEREB200能够结合到ZmEREB180启动子上。综合以上分析，初步建立了ZmEREB180介导的渍水胁迫调控网络。此外，利用耐渍性强自交系A3237和渍水敏感自交系A3239杂交构建F2分离群体，结合候选基因表达和蛋白水平的差异，定位并克隆了耐渍候选基因GRMZM2G179981，验证了其可用于耐渍性遗传改良的优异单倍型。同时，对二叶一心期B73根系进行渍水处理，取渍水处理前、处理后2h、4h、6h、8h、10h和12h根尖进行RNA-Seq分析，共鉴定到3190个差异表达的lncRNA，这些差异表达的lncRNA显著富集到渍水胁迫响应的关键信号通路中。差异表达的lncRNA与蛋白编码基因表达量以及存活率均存在显著关联，且接近59%的差异表达的lncRNA与前人鉴定到的QTL共定位，表明差异表达的lncRNA也影响玉米苗期耐渍性。通过本项目的开展，不仅克隆了耐渍候选基因，也丰富了渍水胁迫调控网络，为耐渍性遗传改良提供了可用的基因资源。