一个玉米干旱敏感突变体基因的图位克隆与功能分析

It is important and useful to study and understand the molecular genetic mechanism of maize in response to drought stress and to discover the key genes. Recently we have identified a novel maize drought-sensitive mutant ds, which was genetically regulated by a single recessive gene. Now we have mapped this gene on the maize chromosome 3 within a 5 Mb interval by the map-based cloning strategy. Based on the above results, in this program larger genetic population will be used to fine map and clone the target gene. Then we plan to confirm the function of DS gene by transgenic complementation experiment and screening new allele mutant, to test the gene function to drought stress by transgenic maize plants, to survey the morphological and physiological characteristic of ds mutant under drought stress, to observe the subcellular localization by GFP fusion protein and analyze expression profiles of DS gene during abiotic stresses by RT-PCR method, to study its molecular regulation pathway using maize genome arrays. We believe that this research will help us to understand how the DS gene respond the drought stress in maize, and the results will be useful in the maize breeding to improve crop stress tolerance.

解析玉米响应干旱胁迫的分子遗传调控机制，发掘其调控关键基因，具有重要的理论意义和应用价值。我们新发现一个玉米干旱敏感突变体ds，该突变体表现为对水分胁迫非常敏感，表现为单基因隐性遗传。目前，已经通过图位克隆的方法将目的基因定位于玉米第3号染色体短臂上，物理距离为5 Mb的区间。本项目拟在此基础上，进一步扩大遗传群体精细定位目的基因，并最终克隆到DS基因。通过转基因互补实验、筛选新的等位突变体等方法确证DS基因的功能；进一步利用转基因玉米检测DS基因的抗逆功能及实用价值；测定ds突变体在干旱胁迫下生理生化指标的变化；运用RT-PCR、GFP融合蛋白等方法分析DS基因在玉米干旱胁迫中的表达及亚细胞定位特性；利用RNA-Seq表达谱分析研究LS基因所参与的分子调控途径。研究结果将有助于进一步解析DS基因参与玉米响应干旱胁迫的遗传调控机制，将为培育抗旱玉米新品种提供新的途径和理论。

利用我们新发现的玉米干旱敏感突变体ds，扩大遗传定位群体，通过图位克隆的方法，精细定位目的基因，最终克隆到DS基因。通过转基因互补实验和目的基因CRISPR敲除实验确证了DS基因的功能。在新疆大田干旱的条件下，发现突变体ds的产量相对于背景材料显著降低。大田条件下突变体的光合速率、蒸腾速率、气孔导度都显著下降，叶片中的ABA含量在午后显著升高，扫描电镜发现突变体的气孔开度明显下降。在温室条件下，检测突变体ds干旱后的存活率显著下降。通过注射烟草表皮细胞实验，发现DS蛋白定位于细胞核、细胞质和细胞膜上。基因表达谱分析结果显示，受环境刺激反应的基因，尤其是水分胁迫反应基因在突变体ds中显著富集。研究结果将有助于科学家深入了解玉米响应干旱胁迫的分子遗传调控机制。