玉米耐渍QTL qRRDW1的功能解析

Waterlogging stress is increasingly becoming one of the major constraints on maize production and productivity. We analyzed 378 maize inbred lines by GWAS analysis on 39,797 SNPs and six traits, including relative root dry weight. Under waterlogging stress, thirteen QTLs associated with relative root dry weight were identified. QTL qRRDW1, located on chromosome 5, stands out as the most significant one associated with relative root dry weight among the thirteen QTLs. The qRRDW1 locus encompasses 50 Kb containing three genes. Among the three genes, ZmWST1, whose mRNA expression is significantly increased upon waterlogging, cold, and NaCl stresses, encodes a calcium-binding protein. An Arabidopsis mutant of the ZmWST1 homolog is sensitive to waterlogging stress. These results suggest that ZmWST1 is a strong candidate associated with waterlogging tolerance. Here, we propose to confirm the ZmWST1 candidate gene and identify the favorable ZmWST1 allele that confers tolerance. We will generate transgenic maize lines that either overexpress the favorable ZmWST1 allele (ZmWST1ox) or carry ZmWST1-knockout generated through the CRISPR/Cas9 technology. The ZmWST1ox and ZmWST1-knockout plants will be tested for waterlogging tolerance, and several traits will be evaluated, including relative root dry weight, ADH1 and POD activities, and GA, ABA, ACC, and SA contents. We will also assess their mRNA and small RNA transcriptomes under normal and waterlogging stress conditions. These studies will unravel the molecular mechanism underlying ZmWST1-mediated waterlogging tolerance to maize, significantly contributing to the understanding of maize response to waterlogging stress.

涝渍已成为限制玉米生产的主要非生物胁迫因子之一。课题组通过GWAS分析，鉴定出13个位点与涝渍胁迫下相对根干重关联。最显著关联位点qRRDW1位于第5染色体，该位点50 Kb区段内有3个编码基因，其中ZmWST1是钙结合蛋白编码基因。涝渍、NaCl、冷胁迫处理，ZmWST1的mRNA表达水平显著升高；拟南芥ZmWST1同源基因突变体，耐渍能力明显降低。由此推断，ZmWST1是qRRDW1位点的耐渍候选基因。本研究拟发掘ZmWST1的优异等位基因；获得转优异等位基因的ZmWST1ox植株、ZmWST1优异等位基因功能丧失的编辑植株；分析ZmWST1ox植株、ZmWST1功能丧失编辑植株的根系形态与结构，根系ADH1、POD等酶活性，激素含量，全转录组表达水平等，以及这些性状在涝渍胁迫下的变化；以期阐明ZmWST1的分子作用机制。本研究将为揭示玉米耐渍性形成的生物学基础积累有益的资料。

在我国南方地区，涝渍胁迫已成为限制玉米生长的主要环境因子之一。本研究通过GWAS技术，发掘了玉米耐渍关键基因ZmWST1，该基因编码钙调素结合蛋白；亚细胞定位显示ZmWST1蛋白作用于细胞核中。在苗期，ZmWST1在不定根的mRNA水平显著高于侧根。ZmWST1突变导致玉米生育期延迟，其中，主要影响拔节期；相对于WT，ZmWST1-KO拔节期延迟了近8 d；此外，ZmWST1-KO籽粒的粒长、粒宽显著降低。与WT相比，正常及涝渍胁迫下，ZmWST1-ox的不定根数量显著增多；相反，ZmWST1-KO的不定根数量显著减少。组织切片显示，ZmWST1-ox不定根根原基形成较早，而ZmWST1-KO较迟。ZmWST1-GFPox为材料，进行DNA-chip分析，结果发现ZmWST1蛋白与1098个DNA位点结合。已报道玉米根系发育相关基因有22个，DNA-chip数据显示，ZmWST1蛋白均不与22个基因启动子结合。RNA-seq数据显示，正常条件下，相对于WT，ZmWST1-KO根系中上、下调表达基因数分别为512、586。综合DNA-chip、RNA-seq结果，我们推断ZmWST1蛋白可能与RING1、NRT1、WRKY51启动子结合。qRT-PCR结果证实，RING1、NRT1、WRKY51在ZmWST1-KO根系的转录水平显著降低。酵母单杂交结果证实，ZmWST1蛋白结合RING1、NRT1、WRKY51启动子。基于以上研究，我们提出了ZmWST1的分子作用机制：钙调素CaM识别植物细胞发出的Ca2+信号，并与ZmWST1结合，然后激活RING1、NRT1、WRKY51的mRNA水平。激活后的RING1、NRT1、WRKY51调控不定根相关基因的表达，从而调节玉米的耐渍能力。本研究为揭示玉米耐渍性形成的生物学基础积累有益的资料，为玉米耐渍性遗传改良提供了新的基因资源。