一个新的赤霉素敏感型棉花矮化突变体的精细定位及其候选基因鉴定

Cotton dwarf mutants are very important for illuminating the mechanism of shoot apical meristem development, high yield breeding and machine-picked cotton production. We found a novel gibberellin-responsive (GAs) dwarf mutant AS98 in cotton which was controlled by a single dominance gene，and was coarsely located on chromosome 25th (Dt6). To clone and clarify the molecular mechanism of the dwarf mutant genes, we will carry out the following research: Based on the genome sequencing information of upland cotton, we will excavate new molecular markers such as SSR,SNP etc.,improve the density of the molecular makers closed to the targeted gene on chromosome 25th, and fine map the dwarf gene by the map-based cloning techniques ；And then the candidate dwarf gene related to GA synthesis adjacent to targeted gene regions will be selected and cloned, and after analyzing the differentiation of gene structure, the yeast hybrid system will be used for making clear if there is a transcriptional regulation between the dwarf gene and the GA synthesis related genes; The variation of expression pattern of the candidate genes will be revealed in the mutant and its wild type after the exotic GA is used , meanwhile the virus-inducing gene silencing (VIGS) technique will be employed for validation the candidate gene functions. It predict this study will acquire the candidate genes closely related to dwarf mutation, and discover the relationship between the dwarf mutation and the GA synthesis genes. This project is significant to reveal the dwarfing mutation mechanism, establish the theory and technology for dwarf plant breeding, and finally realize the high yield with high efficiency in cotton production.

棉花矮化突变体在阐明棉花茎尖生长发育机制、高产育种和机采棉生产中具有十分重要的作用。本研究团队前期研究发现一个赤霉素缺陷型矮化突变体AS98，受一对显性基因控制，并初步定位在第25 号（Dt6)染色体上。为了克隆矮杆基因，明确矮化突变的分子机理，将主要开展如下研究：基于陆地棉基因组序列信息开发新的SSR、SNP等分子标记，加密遗传图谱，精细定位矮杆基因；筛选和克隆目标区间内与GA合成相关的矮杆基因, 并进行基因结构差异分析，利用酵母杂交系统明晰矮杆基因与赤霉素合成是否存在转录调控关系；分析突变体和野生型外施赤霉素后候选基因的表达模式变化，利用病毒诱导的基因沉默(VIGS）等技术验证候选基因功能。该研究预期可获得与棉花矮化突变紧密相关基因，并阐明其矮化是否与赤霉素合成有关。该项目对揭示植物矮化机理，建立矮杆育种的理论和技术，实现棉花高产高效都具有较大意义。

提高棉花产量是棉花育种者的主要任务，相较于提高单株产量，培育具有适合高密度种植（high planting density, HPDA）的栽培品种对提高棉花产量具有更大的潜力。因此，发掘和鉴定适宜密植的棉花种质资源将有助于加快棉花的株型育种进程，进而提高棉花的产量。该研究以一个棉花天然矮化突变体AiSheng98（AS98）为研究对象，它具有株高矮、分支短和分支角度小等适宜密植的理想株型特征。为阐明AS98矮化机制，我们利用QTL-seq检测到D12染色体上一个长度~13.5kb的串联重复，该串联重复的发生引起了AS98突变体D12染色体上的GhDREB1B（Dehydration Responsive Element Binding factor）基因拷贝数加倍，导致该基因的表达量显著升高，从而影响了AS98的株型。随后利用VIGS（病毒诱导基因沉默）技术干涉GhDREB1B基因表达，可以一定程度上恢复AS98株高。相反，在棉花品种R15中过量表达GhDREB1B基因，转基因棉花植株变矮、果枝变短、果枝夹角变小，证明GhDREB1B基因是导致AS98棉花株型矮化和紧凑的关键基因。进一步研究发现，AS98棉花中GhDREB1B基因的上调表达主要影响了赤霉素、生长素信号途径，同时也改变了多个乙烯和油菜素内酯信号途径的基因表达。农艺性状分析发现，转基因棉花的植株矮化程度和GhDREB1B表达水平密切相关，但对纤维发育的影响却较小。因此，通过调控GhDREB1B表达水平可以实现对棉花株高、果枝长度和夹角等株型调节，从而适宜于密植。该研究为培育适宜于密植的棉花育种提供了重要的基因资源和理论指导。