小麦低分子量麦谷蛋白基因座位结构、变异与功能优化的研究

Low-molecular-weight glutenin subunits (LMW-GSs) are among the major components of wheat storage proteins, and play an important role in determining the bread-making quality of bread wheat. However, LMW-GSs display high polymorphic protein complexes encoded by multi-gene family, and elucidating this gene family in bread wheat remains challenging, although huge efforts have been invested. Previously we identified all the LMW-GS genes in Xiaoyan 54 and attributed its superior dough quality to the large number of expressed LMW-GS genes at Glu-A3 and Glu-D3 loci. Further, we developed a marker system and a gene cloning strategy for efficiently identifying all the LMW-GS genes in bread wheat. Moreover, we got the draft genome sequence of wheat A genome progenitor Triticum urartu, and constructed its physical map. In this work, to dissect the LMW-GS loci and understand their gene structure, we will screen the BAC library of Triticum urartu representative line G1812 using the available information on LMW-GS genes, and the draft genome and physical map of G1812. All the BAC clones (Expected to be 2000 kb) within the LMW-GS loci will be selected for separate shotgun sequencing through a minimal-tiling-path strategy and the sequences will be assembled with the software SOAPdenovo. The assembling will be analyzed with the software Fgenesh, GeneMark, PLACE, DotPlot, Repeat Masker and PlantCARE for gene mining, repetitive elements and retrotransposon identification. All the genes (e.g. LMW-GS genes, gliadin genes and powdery mildew resistance genes) and elements will be investigated to reveal the genetic composition and structural variation of the LMW-GS locus. Subsequently, the physical map of Glu-A3, B3 and D3 in bread wheat will be constructed, and the corresponding clones will be shotgun sequenced and assembled. All the sequence data of the LMW-GS loci in T. urartu and T. aestivum will be thoroughly compared to characterize the structural variation and evolutionary clues, which will model the gene composition, regulatory elements and expression patterns of this important locus. The insight into the LMW-GS loci will greatly improve the developed LMW-GS gene marker system, which will be used to survey the composition of LMW-GS genes and their allelic variants in newly released wheat varieties in China. The different performance of LMW-GS gene variants in dough quality will be analyzed by the association study. This study will provide elite gene sources for breeding new varieties with better dough quality and speed up the molecular breeding of superior quality wheat.

小麦低分子量麦谷蛋白（LMW-GS）是小麦籽粒贮藏蛋白的主要组成部分，对营养品质和面粉加工品质有着重要影响，但因其编码基因多且相似度高，难以有效地分离和鉴定。本研究在建立了小麦LMW-GS基因克隆方法和鉴定体系、小麦A基因组框架图和物理图谱的基础上，通过对乌拉尔图小麦代表性品系LMW-GS基因座位（2000kb以上）的测序和序列组装，明确整个基因座位中基因和转座子的结构特征及其调控元件的组成，深度解析乌拉尔图小麦LMW-GS基因座位及结构变异；构建并获得普通小麦完整的LMW-GS基因座位信息，分析不同倍性小麦LMW-GS基因结构特征及进化关系，阐明其基因组成、调控元件及表达调控规律。通过完善小麦LMW-GS基因鉴定体系，明确我国主要优质小麦品种的LMW-GS组成，关联分析LMW-GS基因等位变异的功能差异，为小麦品质育种提供新的育种元件，推动优质小麦新品种的培育和产业发展。

小麦低分子量麦谷蛋白（LMW-GS）是小麦籽粒贮藏蛋白的主要组成部分，对营养品质和面粉加工品质有着重要影响，但因其编码基因多且相似度高，难以有效地分离和鉴定。本研究在建立了小麦LMW-GS基因克隆方法和鉴定体系、小麦A基因组框架图和物理图谱的基础上，对157份来自“新月沃地”的乌拉尔图小麦材料进行了LMW-GS基因的扫描和克隆，获得了这些材料中LMW-GS基因的变异情况；对部分材料进行2-DE和质谱鉴定；利用特异引物对乌拉尔图小麦品系PI 428198的BAC文库进行筛选，得到了由39个阳性单克隆组成的重叠群，对其包含的39个阳性单克隆进行了PacBio测序，拼接成了3.49 M的序列，明确其基因组成和结构特征，并对其包含的储藏蛋白基因的启动子区的顺式作用元件进行了分析；同时对PI 428198连续灌浆期的胚乳做了转录组分析，明确了所有储藏蛋白的表达模式，共表达分析确认可能30类共86个转录因子参与乌拉尔图小麦LMW-GS基因表达调控。对中国春的基因组Glu-A3、Glu-B3和Glu-D3座位序列进行了分析，分析不同时期的转录组，明确了普通小麦中国春Glu-3基因的表达模式；并对218份粗山羊草Glu-D3位点的基因组成进行分析，利用双向电泳以及质谱分析对粗山羊草Glu-D3位点表达的基因进行了鉴定；对来自乌拉尔图小麦、粗山羊草和普通小麦3个亚基因组的Glu-3位点进行了共线性分析。选取包括中国主栽品种、重要亲本、地方品种及外国引进品种的2787份重要小麦种质材料，利用LMW-GS基因分子标记鉴定体系，对这些小麦品种中的LMW-GS基因组成进行了分析。该研究明确了整个基因座位中基因和转座子的结构特征及其调控元件的组成，深度解析乌拉尔图小麦LMW-GS基因座位及结构变异；构建并获得普通小麦完整的LMW-GS基因座位信息，分析不同倍性小麦LMW-GS基因结构特征及进化关系，阐明其基因组成、调控元件及表达调控规律。为小麦品质育种提供新的育种元件，推动优质小麦新品种的培育和产业发展。