矮秆波兰小麦矮化基因Rht-dp的精细定位、克隆和功能分析

Different mechanisms of plant dwarf or semi-dwarf genes, primarily involved in plant phytohormone biosynthesis, transport and signaling, have been/being studied and discovered. In addition to these phytohormone-related dwarfing genes and mechanisms, several dwarfing genes, unbalanced lignin or flavonoid metabolisms, also cause dwarfisms in rice, tobacco, and Brachypodium distachyon. In wheat, more than 20 dwarfing genes were discovered and mapped. A few of dwarfing genes, involved in GA biosynthesis, transport or signaling, were studied. Other dwarfing genes and their mechanisms are unknown. Transcriptomic and proteomic analysis revealed that Dwarf polish wheat dwarfing genes, Rht-dp mapped on 4BS, were involved in lignin, flavonoid, SAM, SAH and cellulose metabolisms, which was different from dwarfing genes caused the unbalance of GA metabolism. However, the sequence of Rht-dp, and its molecular function and physiological mechanism are still unknown. Based on our previous studies, we will perform fine mapping and targeted next generation sequencing to clone Rht-dp, and use transgenic technology, metabolomics, western blot, qRT-PCR, yeast double hybridization and histochemical straining to reveal its molecular and physiological mechanisms.

植物中具有大量不同的矮化基因及其矮化机制。在水稻、拟兰芥和小麦等植物中的矮化基因主要导致植物体内的激素代谢失衡，阻碍细胞分化和伸长，从而导致矮化。也有少部分的矮化基因引起植物木质素或类黄酮的代谢失衡导致矮化。小麦中发现和定位了超过20个矮化基因，很少矮化基因的矮化机制被了解，且主要引起赤霉素代谢失衡导致矮化。DPW的矮化涉及茎秆伸长期木质素、类黄酮、SAM、SAH和细胞纤维素等合成途径的某些蛋白，不同于赤霉素合成途径，但该矮化基因及其生理和分子机制未知。本研究利用精细定位、目的区域捕获测序、图位克隆、代谢组学等方法相结合，分析分离DPW的Rht-dp矮化基因，利用转基因功能验证、蛋白质杂交、基因定量表达、酵母双杂交、组织化学染色等方法了解其分子机制以及生理机制。

来源于我国新疆吐鲁番地区的矮秆波兰小麦（dwarf Plish wheat, DPW; Triticum polonicum L., AABB）拥有矮化、长粒以及富集铁锌等优良性状，是普通小麦遗传改良的重要基因资源。然而，控制其矮化性状的矮秆基因至今未知。本项目通过对矮秆波兰小麦矮化基因Rht-dp的定位、克隆、转录组学矮化相关差异基因的表达分析以及在不同四倍体小麦中的单倍型分析，揭示了Rht-dp为“绿色革命”基因Rht-B1b，该基因降低株高效应能够被PH-TPW部分抑制；该基因通过影响激素、抗氧化活性和氮代谢相关途径，修饰细胞壁结构和抑制细胞壁松弛，从而抑制细胞伸长引起DPW矮化。单倍体分析表明Rht-B1b可能来源于波兰小麦。Rht-dp与Jianyangailanmai的Rht22在降低株高效应上呈现加性效应。通过构建高密度遗传图谱以及新开发SSR标记，将Rht22的遗传候选区域确定在7AS染色体的两个SSR标记之间，为后期该基因的精细定位和图位克隆奠定了物质和理论基础。同时，利用DPW和Jianyangailanmai的重组自交系优良单株与普通小麦主要栽培品种杂交，创制了一系列半矮化、籽粒大和千粒重高的优良遗传材料，为普通小麦的遗传改良奠定了坚实的物质基础。此外，既然Rht-dp的目的基因为Rht-B1b，该基因显著降低氮素的吸收和利用效率。氮肥作为作物生产上使用的大量营养物质，能够有效调控作物对营养元素和有毒金属元素的吸收和积累。因此，项目组进一步揭示了Rht-dp背景下不同氮素形态调节矮秆波兰小麦生长和金属元素积累的生理和分子机理。该项目的完成证据充分地揭示了矮秆波兰小麦矮化基因Rht-dp的目的基因；深入地阐述了采集于中国新疆吐鲁番地区的矮秆波兰小麦矮化的生物学基础；为后期Rht22基因的精细定位和克隆奠定了理论基础；评价和诠释了Rht-dp背景下氮素调控矮秆波兰小麦的营养元素和有毒金属元素的吸收和积累的生理和分子基础；创制了一系列半矮化以及粒大和千粒重高的优异育种材料，为普通小麦矮化、产量和品质育种奠定了坚实的物质基础。

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