玉米粒重主效qGW1.04的图位克隆及功能解析

Grain weight is a direct component of maize yield, and it is important to analyze the function of gene for grain size. In our previous research, a major QTL for grain weight, qGW1.04, was fine-mapped to an 198.3 kb region on chromosome 1 using a large BC5F2 population derived from the cross between the inbred line N04 with small grain (100-grain weight, 9.15±0.18 g) and the inbred line Dan232 with large grain (100-grain weight, 21.06±0.22g). Three genes were identified in this region. Further researches in this study are as follows: (1) to development within the target range of new molecular markers associated with screening of recombinant cDNA and different grain weight per plant material of candidate genes and the promoter sequence analysis to determine the candidate genes qGW1.04; (2) functional analysis of qGW1.04 candidate gene through RNAi interference and CRISPR-Cas9 edit; (3) qGW1.04 candidate gene subcellular localization and in situ hybridization analysis of candidate gene expression period, as well as parts of the expression regulatory role in cell number and cell size; (4) RNAi-seq analysis of endosperm and peel at different periods in Near-isogenic lines and N04, and yeast two-hybrid system were used to screen the interaction of protein molecules. The molecular mechanism of qGW1.04 candidate gene was regulated by grain weight. The mechanism and regulation mechanism of qGW1.04 candidate gene were comprehensively analyzed to provide a new basis for improving yield.

粒重是玉米产量的重要构成因素之一，研究粒重相关基因功能具有重要应用价值。课题组利用玉米小粒自交系N04与大粒自交系丹232杂交构建分离群体，将百粒重主效QTL，qGW1.04精细定位在包含3个基因的198.3kb区段内。在此基础上，本项目拟开展以下研究：（1）目标区间内开发新的分子标记筛选重组单株及不同粒重材料候选基因cDNA和启动子序列关联分析，确定qGW1.04候选基因；（2）qGW1.04候选基因的RNAi干扰及CRISPR-Cas9编辑玉米遗传转化功能分析；（3）qGW1.04候选基因亚细胞定位及原位杂交，分析候选基因表达时期、表达部位以及对细胞数目和细胞大小的调控作用；（4）近等基因系、N04授粉后不同时期胚乳和果皮RNA-seq分析以及酵母双杂交系统筛选互作蛋白分子，分析qGW1.04候选基因调控籽粒粒重的分子机理。综合解析qGW1.04候选基因的功能及调控机制，为改良粒重提高产量提供新依据。

粒重是产量的直接组成因素，挖掘控制粒重的相关基因揭示其功能具有重要意义。项目组利用普通玉米自交系丹232和小粒爆裂玉米自交系N04构建F2:3、BC2F2、RILs群体定位百粒重QTL于1号染色体基础上，构建回交群体精细定位控制粒重主效基因，并解析其调控粒重的分子机理。主要研究进展包括三个方面：（1）构建近等基因系精细定位到粒重QTL qGW1.04主效基因ZmGW1，ZmGW1编码SINA家族蛋白。自然群体自交系关联分析表明ZmGW1变异为N04和Dan232两种单倍型，启动子Indel-1345等位变异与粒重紧密连锁。启动子活性及基因表达模式分析Indel-1345增强ZmGW1表达负调控粒重与籽型；（2）近等基因系不同发育时期细胞学分析及储藏物质含量测定表明，ZmGW1影响胚乳细胞发育及胚乳灌浆。过表达和CRISPR基因编辑遗传验证了ZmGW1负调控粒重与粒型；（3）ZmGW1为具有核定位信号的自主泛素化E3泛素连接酶。近等基因系转录组分析表明ZmGW1在mRNA水平影响了碳水化合物及小分子合成与代谢。泛素化蛋白质组分析富集到被泛素化差异蛋白主要为脂质储藏，蛋白泛素化途径。酵母双杂交文库筛选ZmGW1互作蛋白同样筛选到泛素化途径蛋白，包括SINA家族其他蛋白。综合上述结论，我们图位克隆到一个同时控制普通玉米和爆裂玉米的主效基因ZmGW1，初步解析其调控粒重与粒型的分子机理，为玉米产量性状遗传改良提供重要的理论依据。