玉米叶夹角形成的分子机理研究

Leaf angle is one of important agronomic traits affecting the yield in maize. Upright leaves can significantly improve photosynthetic efficiency and increase planting density, and then increase maize yield. The quantitative trait loci (QTL) for leaf angle were mapped using three sets of F2:3 families in our previous study. Three major QTL (qLA1, qLA2, qLA4) were detected and the corresponding near-isogenic lines were constructed. The three QTL had been fine mapping. At present, the candidate gene in the qLA1 region had been cloned and its function was validated by association analysis. On this basis, the researches aim to clone the candidate genes of qLA2 and qLA4 regions by map-based cloning. the functions will validated by association analysis and genetic transformation. The expression pattern, interaction proteins and transcription factors of three candidate genes will be studied by proteomic techniques and the protein interaction network will be constructed, which will suggest signal transduction of leaf angle formation. Differentially expressed genes will be identified and the relationships between the three candidate genes and new identified genes will analyzed by the transcriptome sequencing. The action mechanisms of leaf angle formation will identified from the cellular level. The implementation of this project will provide a theoretical basis not only for the corn resistant to the dense high-yield breeding and molecular marker-assisted selection but also reference for biological functions of key genes and protein in the signaling interaction network during the formation of leaf angle.

玉米叶片夹角是影响产量的重要农艺性状，直立的叶片可显著提高光合效率、增加种植密度，进而增加产量。课题组利用3套F2:3家系对叶夹角进行QTL定位，对检测到3个主效QTL qLA1、qLA2、qLA4构建近等基因系并进行精细定位。已克隆到qLA1的候选基因，并通过关联分析初步验证其功能。在此基础上，本研究拟通过图位克隆技术克隆qLA2和qLA4的候选基因，利用遗传转化和关联分析方法验证其功能；利用蛋白质组学技术研究3个候选基因的表达规律，发掘与其互作的蛋白质和相应的转录因子，构建蛋白质互作网络，分析叶夹角形成的信号转导途径；利用转录组测序技术鉴定具有差异表达的新基因，解析3个候选基因与新鉴定基因的关系；从细胞水平上分析叶夹角形成的作用机制。本项目实施不仅对玉米耐密高产育种及分子标记辅助选择提供理论依据，而且对解析叶夹角形成过程中关键基因及蛋白信号在互作网络中调控的生物功能提供参考。

叶夹角是决定玉米耐密特性的一个重要性状，解析玉米叶夹角形成的分子机制，不仅具有重要的科学价值，而且对于选育耐密高产玉米新品种具有重要的实践意义。课题组利用紧凑型自交系豫82 为供体，以松散型自交系沈137、豫87-1、D132 为受体，采用回交方法构建qLA1、qLA2、qLA4 NILs 并进行精细定位。图位克隆了qLA1、qLA2、qLA4的候选基因，分别命名为CLA1、CLA2、CLA4。关联分析结果表明，在CLA1启动子区域共检测出26个多态性位点与叶夹角存在显著关联，其中InDel-1094和InDel-1109改变了序列的二级结构，与其它多态性位点存在成因关系，进而影响其它位点对叶夹角的影响；ZmCLA2基因3'-UTR的3个多态性位点C/T/mutation57 、 ACT/indel64和CGCA/indel283与叶夹角之间存在显著关联；ZmCLA4 基因的cDNA序列中C/T/mutation607 和 CA/indel965与叶夹角之间存在显著关联。转基因及qRT-PCR结果表明，ZmCLA1是正向调控叶夹角大小的关键基因； ZmCLA2、ZmCLA2负向调控玉米叶夹角的大小。细胞学分析结果表明ZmCLA1基因可能通过影响细胞的发育从而调控玉米叶夹角；ZmCLA2通过影响维管组织附近的机械支撑组织的细胞层数从而调控玉米叶夹角；ZmCLA4可能影响细胞分裂和发育从而导致叶夹角的变化。通过酵母双杂交、RNA-Seq、ChIP-Seq分析结果表明，ZmCLA1作为两种植物激素（BR和IAA）信号传导路径的交叉节点调控叶夹角的大小,即通过抑制BR信号传导路径中CLA2的表达抑制MAPK的高效表达影响叶枕部位远轴端厚壁细胞层数减少，机械组织不发达，促使叶夹角的增大；同时CLA1抑制IAA17的表达、CLA4促进IAA17的表达，进而影响生长素的极性运输，导致叶夹角的改变。蛋白质组学结果表明，ZmCLA1通过影响糖蛋白、PER、LOX等木质素合成与降解相关的蛋白质，影响玉米叶片夹角的大小。此外，利用qLA1位点开发的功能标记创制的紧凑耐密型自交系组配出了豫单9953和豫单903分别通过了国家级和省级区相关试验，达到了审定标准。这些研究结果为解析叶夹角形成的分子机制及在分子标记辅助选择育种创制优异种质提供参考。