玉米无雄穗基因vt3的克隆及功能分析

Meristems maintenance and differentiation are responsible for the plant growth and development, studies on meristem mutants are an important means of analyzing the mechanism of plant organogenesis. Using heavy-ion radiation, we screened a daylength/temperature-dependent tassel-vanishing mutant vt3 (vanishing tassel3), the mutant can develop normal male inflorescence when sowing in the spring of Changsha, the summer of Beijing and the winter of Hainan; however, the mutant can show the phenotype of completely vanishing tassel when planted in the autumn of Changsha. Through map-based cloning and sequencing analysis, we determined the candidate gene of vt3. In this project, we will ascertain the daylength/temperature mechanism of vt3 mutant via indoor simulating experiment; verify the candidate gene using transgenic technology; investigate the expression pattern of VT3 gene by RT-PCR, qRT-PCR and mRNA in situ hybridization; determine the localization and function of VT3 protein by means of subcellular localization and prokaryotic expression; analyze the genetic networks of VT3 gene by RNA-SEQ analysis; And then provide insight into VT3 gene function in regulating the initiation of maize male inflorescence meristem. Finally, combined with the daylength/temperature mechanism of vt3 mutant and functional analysis of VT3 gene, we will uncover a novel molecular mechanism of the initiation of maize male inflorescence meristem coordinately regulated by genes and daylength/temperature.

分生组织维持与分化决定了植物的生长和发育，对分生组织突变体进行研究是解析植物器官形成机理的重要手段。申请者前期通过重离子辐射筛选了一份依赖于光温条件的玉米无雄穗突变体vt3（vanishing tassel3），该突变体在长沙春播、北京夏播和海南冬繁时可发育形成正常的雄穗，而在长沙秋播时可表现出雄穗完全缺失。申请者利用图位克隆和测序分析确定了vt3的候选基因。本研究拟通过室内模拟试验探明突变体的光温机制；应用转基因技术验证候选基因；采用RT-PCR、qRT-PCR和原位杂交考察目的基因的表达模式；通过亚细胞定位和原核表达明确目的蛋白的定位和功能；利用转录组测序挖掘目的基因可能的调控网络；进而解析VT3基因在调控雄穗花序分生组织起始中的功能。最后，结合vt3的光温机制和VT3的基因功能分析，揭示基因与光温条件共同调控玉米雄穗花序分生组织起始的分子机制。

温度是调节植物生长和发育的重要环境因子。为了在极端温度下存活并繁殖后代，植物必须响应温度变化以保证花器官的正常形成和发育；然而，相应的分子机制仍待解析。在本研究中，我们鉴定了一个玉米温敏无雄穗突变体thermosensitive vanishing tassel1-R（tvt1-R；项目申请书中将其指定为vt3），其在限制性高温下由于顶端分生组织（SAM）败育表现出雄穗缺失，而在许可性适温下雄穗正常。tvt1-R突变体雄穗表型转换的关键时期是V2-V6（Vn，此处‘n’表示可见叶舌的叶片数）。图位克隆、等位测验及互补测验证明核糖核酸还原酶（RNR）大亚基（RNRL）基因ZmRNRL1上一个导致其编码蛋白第277位精氨酸（Arg277）替换为组氨酸（His277）的G-A点突变赋予了tvt1-R突变体的温敏无雄穗表型。RNR调节DNA复制和损伤修复所需脱氧核糖核苷三磷酸（dNTP）的合成速率。通过基因表达、酵母双杂、RNA-seq及流式细胞等分析，我们揭示了一个限制性高温下玉米雄穗缺失的分子机制：在较高的温度（34℃）下，ZmRNRL1-tvt1-R的Arg277-His277替换影响了它与3个RNR小亚基（ZmRNRS1、ZmRNRS2和ZmRNRS3）的互作，这将影响RNR全酶的形成，造成DNA复制所需的dNTP供应不足；这种影响对需要持续充足dNTP供应进行快速分裂的SAM尤为严重，进而导致了SAM败育和雄穗缺失。这一研究为核苷酸稳态在SAM维持中的作用提供了新见解。