调控玉米叶夹角的关键基因CLA7-1的遗传网络解析

Improving plant architecture, increasing planting density and photosynthetic efficiency are important ways to achieve high yield breeding in maize. Leaf angle is one of the most important characters of maize plant architecture. Previous studies showed that brassinosteroid signaling pathway plays an important regulatory role in the development of maize leaf angle. In this study, we cloned ZmCLA7-1 through map-based cloning, which is responsible for the BR signal transduction pathway in the control of maize leaf angle, and the key genes that interact with it were investigated using yeast hybridization, CHIP-Seq and co-immunoprecipitation assays to construct interacting networks; Transcriptome analysis were applied by using knock-out mutant from CRISPR-Cas9 mutagenesis. Furthermore, BR treatment was applied to the mutants, as to reveal the relationship among CLA7-1, CLA1 and CLA2 in the BR signal transduction pathway and to elucidated the genetic networks and molecular mechanisms of the BR conduction signal pathway in the regulation of maize leaf angle development. This project provides theoretical and technical support for maize high-yield breeding.

以改良玉米株型、增加种植密度、提高群体光合效率为目标的株型育种，是实现玉米高产再高产的重要途径。叶夹角是决定玉米株型特征最重要性状之一，在玉米叶夹角发育中油菜素内酯(BR)信号传导途径发挥着重要的调控作用。本研究在前期图位克隆到控制茎叶夹角发育中BR信号传导途径的关键基因ZmCLA7-1基础上，利用酵母杂交、CHIP-Seq、免疫共沉淀等技术发掘与其互作的关键基因，构建该候选基因的互作网络；利用CRISPR-Cas9技术获得突变为材料通过转录组技术分析候选基因互作网络间基因的调控关系；通过BR处理突变体等材料，综合分析CLA7-1与CLA1、CLA2在BR信号传导路径中的调控关系，初步阐明玉米茎叶夹角发育中BR传导信号路径的遗传网络和分子机制。本项目的实施为玉米高产分子设计育种提供理论及技术支撑。

以改良玉米株型、增加种植密度、提高群体光合效率为目标的株型育种，是实现玉米高产再高产的重要途径。玉米叶夹角的大小是影响玉米密植的主要农艺性状之一。因此，挖掘调控叶夹角发育的关键基因及其分子机制解析对高产密植新品种的选育具有重要的意义，对解析叶夹角发育的分子机制具有重要的科学价值。本研究利用紧凑型自交系Yu82与松散型自交系D132为试验材料，构建了QTL qLA7-1的近等基因系，并通过图位克隆技术克隆了影响叶夹角大小的关键基因ZmCLA7-1，利用过表达和CRISPR-Cas9基因编辑技术，通过玉米遗传转化，证明ZmCLA7-1正向调控玉米叶夹角；以ZmCLA7-1蛋白为诱饵从酵母双杂交文库中筛选与其互作的基因，从中筛选到3个与其互作的基因：actin、Hy和AE7-1，通过双分子荧光互补实验和蛋白免疫共沉淀技术验证了基因actin和AE7-1与ZmCLA7-1在体内体外均存在互作关系；采用DAP-Seq结合RNA-Seq技术证明了ZmCLA7-1通过直接与ARF11、FUT、E3、IAA10、WRKY40、bHLH157、LA1和LA2的启动子结合增强启动子活性而促进这些基因的表达，而与基因AP2-EREBP109/144/172/196/204、ATG12、bZIP4、和LA4的启动子结合抑制启动活性使这些基因的表达量有所下降。利用前期精细定位获得主效QTL位点开发一对SSR功能标记用于分子标记创制了一批优异种质组配了一系列优良组合。其中梦玉369、豫单776通过国家级审定、豫单9966河南省审定；申请/获得国家专利2项，发表学术论文6篇。