【作者】 武丽芬；

【导师】 刘旭； 陈景堂；

【作者基本信息】 河北农业大学 ， 作物遗传育种， 2024， 博士

【摘要】 抽穗期是小麦的重要农艺性状,决定着小麦的区域适应性和产量等,适宜的抽穗期是小麦高产稳产的根本保障。因此,发掘新的小麦抽穗期调控基因,并解析其分子机制对培育适应气候变化的广适性小麦新品种具有重要意义。本研究以小麦偃展4110（YZ4110）EMS诱变突变体库中分离的一个突变体m406为材料,开展了m406抽穗期性状调控基因的定位、克隆与功能研究,主要研究结果如下:1.m406的晚抽穗表型是由隐性基因控制的。通过六年两地对m406突变体农艺性状的调查发现,与野生型YZ4110相比,m406表现为抽穗期延迟,穗长、小穗数、粒长、粒宽和千粒重等产量相关性状显著提高的特点。利用m406与YZ4110和矮抗58（AK58）配制了两个杂交组合,其F₁植株的抽穗期与YZ4110和AK58无显著差异,但与突变体m406的抽穗期表型差异显著,表明m406晚抽穗表型是由隐性基因控制的。对两个分离群体单株抽穗期表型的遗传分析表明,正常抽穗与晚抽穗的单株比例为3:1,进而表明突变体m406晚抽穗的表型是由一个隐性基因或一组紧密连锁的基因控制的。2.m406中存在的大片段缺失导致其晚抽穗等农艺性状的表型变异。利用小麦55K SNP芯片对AK58×m406 F_2:3分离群体的两个极端表型混合池进行基因型扫描,SNP分布图显示m406控制抽穗期的基因被定位于小麦2D染色体短臂末端约80 Mb的区间内,BSE-Seq,RNA-Seq reads分布图及荧光原位杂交（FISH）的实验结果进一步证明m406突变体存在大约47 Mb的大片段缺失。为了精细确定缺失片段的大小,利用新开发的10个分子标记扫描AK58×m406的分离群体,最终将缺失区间缩小到46.58 Mb。缺失区段内包含917个高可信度基因,其中23个基因与开花时间、穗长或籽粒大小有关。因此m406的表型是由多基因缺失造成的。3.缺失区段内的TaFPF1-2D、TaEMF2-2D和Ppd-D1基因具有调控小麦抽穗期的功能。本研究选择了缺失区段内与小麦抽穗期相关的3个基因Traes CS2D02G000600（TaEMF2-2D）、Traes CS2D02G046600（TaFPF1-2D）和Traes CS2D02G079600（Ppd-D1）作为调控m406晚抽穗表型的候选基因进行功能研究。分别敲除TaFPF1-2D、TaEMF2和Ppd-D1基因,敲除株系均表现为晚抽穗。此外,Ppd-D1敲除株系的粒长、粒宽和千粒重显著减少,但TaFPF1-2D和TaEMF2敲除株系的粒长、粒宽和千粒重均显著增加。过表达TaFPF1-2D和TaEMF2-2D基因的株系均表现为早抽穗的表型。综上表明,TaFPF1-2D、TaEMF2-2D和Ppd-D1的缺失共同造成m406抽穗期等农艺性状表型。4.TaFPF1-2D和TaEMF2-2D能促进小麦抽穗。TaFPF1-2D是一个定位于细胞核和细胞质的蛋白,在所检测的组织中均有表达。开花调控关键基因Vrn1-5A和Vrn3在TaFPF1-2D基因敲除株系中呈下调表达,酵母单杂交和原生质体瞬时表达实验进一步证明Vrn1-5A蛋白可以与TaFPF1-2D启动子上P1基序直接结合,激活TaFPF1-2D的表达。此外,荧光素酶互补成像（LCI）和双分子荧光（BiFC）实验表明TaFPF1-2D与Vrn1-5A、TaEMF2-2D和Ppd-D1分别存在物理互作。TaEMF2-2D也是定位于细胞核和细胞质中的蛋白,在所检测的组织中均有表达。Vrn1-5A、Vrn3和TaFPF1-2D基因在TaEMF2基因敲除株系中呈下调表达,LCI和BiFC实验表明TaEMF2-2D与Vrn1-5A和Ppd-D1分别存在物理互作。综上所述,突变体m406在小麦2D染色体短臂上的46.58 Mb缺失是导致其抽穗期等农艺性状变异的原因;敲除缺失区间内与抽穗期相关的基因TaFPF1-2D、TaEMF2和Ppd-D1的转基因株系均能产生晚抽穗表型,过表达TaFPF1-2D和TaEMF2-2D的转基因株系均能促进小麦提前抽穗。Vrn1-5A能直接结合TaFPF1-2D启动子的P1基序,激活TaFPF1-2D表达促进小麦抽穗。本研究为小麦广适品种的培育提供了基因资源与理论基础。更多还原

【Abstract】 Heading date is a key agronomic trait that influences regional adaption and yield of wheat cultivars.A suitable heading date not only enhances the wheat’s adaptability but also serves as a fundamental guarantee for achieving high and stable yields in wheat production.Therefore,the discovery and elucidation of the molecular mechanisms of genes related to heading date and yield are of great significance for breeding new wheat varieties with broad adaptability to meet the demands caused by climate change.In this study,we cloned the wheat heading date gene and explored its regulatory mechanism in the m406 mutant derived from the EMS mutant library of wheat Yanzhan 4110（YZ4110）.The main results are as follows:1.The delayed heading date phenotype of the mutant m406 is controlled by a recessive loci.Through six years and two locations of investigation on the agronomic traits of the m406 mutant,it was found that compared to the wild-type YZ4110,the mutant m406exhibits a delayed heading date along with significantly enhanced traits associated with grain yield,including increased spike length,number of spikelets per spike,grain length,grain width,and thousand-grain weight.Genetic analysis revealed that the hybrid combination of m406 with two varieties,YZ4110 and Aikang 58（AK58）,showed no significant difference in the heading date phenotype of F₁plants compared to YZ4110 and AK58,but a significant difference in the heading date phenotype with the mutant m406.This result indicates that the late heading date phenotype of m406 is controlled by a recessive loci.Further genetic analysis of the phenotypes of two populations of the heading date showed that the ratio of normal and late heading plants was 3:1,indicating that the late heading date phenotype of the mutant m406 is controlled by a recessive gene or a group of closely linked genes.2.The large fragment deletion in m406 leads to the phenotypic variations such as the late heading date agronomic trait.Two mixed DNA bulk pools with two extreme phenotypes from the F_2:3populations of AK58×m406 were scanned using a wheat 55 K SNP chip.We mapped the candidate gene to an approximately 80 Mb region on the terminal of the short arm of the 2D chromosome.Further analyses provided evidences for a chromosomal fragment deletion of approximately 47 Mb within this region using a combination of approaches of BSE-Seq,RNA-Seq and Fluorescence in situ hybridization（FISH）.Next,10 molecular markers were developed to narrow down the deletion region to46.58 Mb which contained 917 high-confidence genes.Among them,23 genes are related to flowering time,spike length,or grain size,thus the phenotype of m406 may be co-regulated by multiple genes.3.The TaFPF1-2D,TaEMF2-2D and Ppd-D1 genes within the deleted region have the function in regulating heading date.We selected three genes,Traes CS2D02G0000600（TaEMF2-2D）,Traes CS2D02G046600（TaFPF1-2D）,and Traes CS2D02G079600（Ppd-D1）,which are associated with wheat heading date,within the deleted region as candidate genes potentially responsible for regulating the phenotype of the mutant m406 for further investigation.By gene editing technique,the knockout mutants of TaFPF1-2D,TaEMF2,and Ppd-D1 were obtained and they showed late heading date.Moreover,the knockout of Ppd-D1 caused a significant decrease in grain length,width and thousand-grain weight,while the knocking out of TaFPF1-2D and TaEMF2 led to significantly increase in grain length,width,and thousand grain weight.While the transgenic overexpression lines of TaFPF1-2D and TaEMF2 showed earlier heading date.Overall,the results suggest that loss of TaFPF1-2D,TaEMF2-2D and Ppd-D1 might caused the late heading date and other associated phenotypes observed in the mutant m406.4.TaFPF1-2D and TaEMF2-2D promote wheat heading date.TaFPF1-2D is a nucleus and cytoplasm located protein.It is expressed in all detected tissues.The key genes for flowering regulation,Vrn1-5A and Vrn3,were down-regulated in TaFPF1-2D knockout lines.Further studies demonstrated that Vrn1 protein can directly bind to the P1 motif within the TaFPF1-2D promoter region and activate the expression of TaFPF1-2D by yeast one-hybrid system and protoplast transient expression experiments.In addition,TaFPF1-2D interacted with Vrn1,TaEMF2-2D,Ppd-D1 by LCI and BiFC assays.TaEMF2-2D is also a nucleus and cytoplasm located protein,and is expressed in all detected tissues.The Vrn1-5A,Vrn3,and TaFPF1-2D genes were down-regulated in TaEMF2 knockout lines.More researches indicated that TaEMF2-2D also interacted with Vrn1-5A and Ppd-D1 by LCI and BiFC experiments.In summary,the 46.58 Mb deletion on the short arm of chromosome 2D in the mutant m406 is responsible for the variation in agronomic traits such as heading date.Knocking out each of the heading date related genes within the deleted interval,namely TaFPF1-2D,TaEMF2,and Ppd-D1,individually leads to a late heading date phenotype,demonstrating their involvement in determining the heading date.Overexpression of each TaFPF1-2D and TaEMF2-2D gene can accelerate the heading date in wheat.This research will provide theoretical basis and gene resources for breeding broader adaptation wheat variety.更多还原