花生矮杆基因dw1的定位与功能分析

Plant height is one of the important agronomic traits that affect the yield of peanut. The dwarf peanuts have the characteristics of lodging resistance, which is a key factor for the regulation of plant architecture and high yield. Our previous studies have found a single recessive dwarf peanut mutant dw1 (dwarf1). Data suggested that plant height was regulated by this gene through GA signaling pathway. dw1 was roughly mapped on chromosome A04 in the physical region of 5.1 Mb by using linkage analysis and BSA analysis methods with the mapping population. This project will continue to further establish the fine physical map of high density by using larger mapping population, and finally clone dw1 through high precision linkage analysis. We will construct of different expression vectors to analyze the subcellular localization of the gene and spatial and temporal expression characteristics, and to reveal the regulation network of dwarf gene and related potential genes by RNA-seq. The role of this gene played in the GA signaling regulatory network will be investigated by using mutants involved in GA synthesis and signaling pathway in Arabidopsis. The yeast two-hybrid technique will be used to identify and validate the interaction proteins, and to analyze the molecular mechanism of peanut plant height regulation. This project will help us to further understand the molecular mechanism of peanut plant height regulation, and lay the foundation for further studies on molecular assisted breeding and the ideotype plant cultivation of peanut.

株高是影响花生产量的重要农艺性状。矮杆花生具有抗倒伏的特点，是花生株型调控和产量提高的关键因素。我们前期研究发现一个由隐性单基因控制的花生矮杆突变体dw1(dwarf1）,该基因可能通过赤霉素（GA）信号途径调控花生株高。通过构建定位群体，开发分子标记，利用BSA等技术将该基因初步定位在A04染色体上5.1Mb的物理区间。本项目将进一步建立高密度精细物理图谱，通过高精度连锁分析，定位并克隆dw1基因；构建不同表达载体分析该基因的亚细胞定位和时空表达特性；比较突变体和其亲本的表达谱，揭示矮杆基因潜在的调控网络；利用拟南芥中的GA合成以及信号途径中的关键基因突变体，研究该基因在GA信号调控网络中的作用；利用酵母双杂交等技术筛选与其相互作用的蛋白。综合分析DW1基因调控花生株高的分子机理。本研究将有助于深入了解花生株高调控的分子机制，为研究花生理想株型建成和分子辅助育种奠定基础。

株高是影响花生产量的重要农艺性状，高产田花生旺长易倒伏。阐明花生矮杆形成的分子机理可以为抗倒伏、矮杆品种培育提供科技支撑，是花生产量提高的关键。基于我们前期发现一个花生矮杆突变体dw1,研究发现该突变体dw1矮杆表型由隐性单基因控制的，矮杆表型是由于赤霉素合成缺陷导致的。本研究构建了2个定位群体，共开发了110个分子标记用于基因定位，利用BSA和初步定位将DW1基因定位于D446标记附近，通过精细定位进一步将该基因定位于0-512 kb区间。该区间一共有48个开放阅读框，基因注释结果显示只有N16（arahy.Y00ULH）与赤霉素的代谢相关，其编码一个类似于二氧化戊二酸依赖的双加氧酶。因此，推测该基因突变后抑制了花生内源GA的合成和信号转导，从而使体内缺乏活性GA，从而导致花生株高变矮。转录组数据表明，arahy.Y00ULH在22个花生组织中的表达量都比较低，而在根和根瘤中的表达相对较高。qRT-PCR结果显示矮生突变体中该基因的表达量显著低于野生型亲本。GC-MS分析发现，与野生型相比，突变体dw1茎中赤霉素GA3的含量下降，合成通路上其他的关键组分GA34，GA20，GA1，GA5的含量均不同程度下降。证实了突变体中基因突变导致赤霉素合成能力下降导致矮杆表型。构建了花生cDNA文库，利用酵母双杂交法筛选到了1个与arahy.Y00ULH互作蛋白。从LA群体高代株系中，筛选到株高在25-40厘米之间的株系35个，且单株结果数大于15的株系12个，经检测这些株系都与矮杆基因dw1连锁。小区试验结果显示，与亲本鲁花14相比有7个株系产量增加10%以上，获得花生新品种登记1项。