小麦DELAYED GERMINATION 1 基因调控种子萌发和穗发芽的分子机理

收获前穗发芽影响小麦产量和质量。鉴定调控种子萌发的关键基因，有助于揭示穗发芽的分子机理，为运用基因编辑技术创制耐穗发芽材料奠定分子基础。基于申请人在水稻中鉴定的正向调控种子萌发的新基因DEG1，在小麦中克隆了功能可能冗余的三个DEG1同源异源基因，运用基因编辑技术创制了三突变体。萌发实验初步表明小麦DEG1基因可能同样调控种子萌发和穗发芽。在获得了突变体等材料和相关实验结果的基础上，项目计划：1)通过萌发和穗发芽等实验，进一步确定基因功能，分析三个基因功能的冗余或差异；2) 通过转录组测序、染色质免疫共沉淀、酵母双杂交等实验鉴定下游基因和相互作用蛋白，解析机理；3) 在分子育种方面开展基础性工作，包括考察突变体产量性状、分析基因多态性。项目围绕穗发芽科学问题，预期在小麦种子萌发和穗发芽的分子机理方面取得突破，为分子遗传改良穗发芽抗性提供具有自主知识产权的候选基因。

Pre-harvest sprouting (PHS) affects the yield and the quanlity of wheat. Identify key genes which regulate seed germination is helpful to reveal the molecular mechanism of PHS, and could lay a molecular foundation for creating materials to resist PHS. Based on our identified new rice DEG1 (DELAYED GERMINATION 1) gene, which regulates seed germination positively, three wheat Homoeologous DEG1 genes were cloned, and triple mutant lines were generated with CRISPR-Cas9 system. Seed germination of triple mutant was delayed compared to the control, implying that wheat DEG1 genes also function in regulating seed germination and PHS, and can be used in genetic improvement of PHS resistance. Based on the generated triple mutant lines, HA tag fused over-expression trangenic wheat lines, expressed and purified GST fused protein, and other materials and gained results, we mke a plan for this porgram: 1) characterize the functions in regulation of seed germination and PHS further, and analyze the functional redundancy or differences between three Homoeologous genes by performing seed germination assay, PHS test and other experiments ; 2) identify downstream genes and interacting proteins of DEG1 transcription factors at genome level to elucidate the molecular mechanism, by performing RNA-Seq, Chromatin Immunoprecipitation Assay (ChIP), Yast two hybrid (Y2H) and CO-IP; 3) do some basic works on molecular breeding: investigate yield traits of triple mutant lines; analyze the polymorphism of wheat DEG1genes in different wheat germplasm resources . This program is focused on PHS, and it is predicted to make significant progress in revealing the molecular mechanism to regulate seed germination and PHS, and provide new candidate genes for genetic improvement of resistance to PHS.