甜玉米种子耐老化基因qGR-1的克隆及功能解析

The kernels become shrunk and low-weight attribute to the restraint of the starch synthesis by the genetic mutation of the endosperm starch synthetic gene or genes in sweet corn, which leads to the difficult of seed storing. Therefore, the cloning of seed aging resistant genes and the unraveling of their molecular mechanisms are the essential conditions for breeding sweet corn cultivars with high seed aging resistance. In the previous study, a joint and stable major QTL on seed aging resistance was identified and explained over 10% of phenotypic variation by two years’ experiments, which was designated as qGR-1 and mapped to the 1.40 cM interval of bin 10.03 including 15 candidate genes. In this project, we will fine-map and clone gene qGR-1 using expanded backcross population, identify the function of qGR-1 by genetic transformation, analyze the characteristic of the expression and network regulation of qGR-1 during seed aging by qPCR and RNA-seq, explore the physiological and biochemical changes of the parental lines and transgenic lines after artificial aging and uncover the molecular mechanism of qGR-1. Moreover, the favorable allele of qGR-1 will be identified based on the genetic diversity analysis using an sweet corn association panel. The functional markers will be developed within gene qGR-1 and the selection effect of the functional markers will be identified by the hybrid progeny from the lines with best allele in qGR-1 and the lines with weak seed aging resistance. This study will not only deepen the understanding of mechanisms on seed aging, but also provide technological support for the genetic improvement of seed aging resistance in sweet corn.

甜玉米因胚乳淀粉合成基因突变抑制淀粉合成，胚乳皱缩，粒重轻，极不易贮藏。因此种子耐老化基因的克隆及分子机制解析是培育耐老化甜玉米品种的首要条件。本实验室前期两年试验鉴定到一个表达稳定的耐老化主效QTL，命名为qGR-1，可解释大于10%的表型变异，位于Bin10.03的1.40cM区间，包含15个候选基因。本项目拟利用扩大的回交群体精细定位并克隆qGR-1基因，利用遗传转化验证基因功能，通过qPCR、RNA-seq等方法鉴定qGR-1调控种子老化的表达及网络调控，分析亲本及转化材料种子老化处理后的生理生化变化，揭示qGR-1的耐老化机理。再结合甜玉米关联群体的遗传多样性分析确定最优等位基因，开发基因内的功能标记，利用最优等位基因自交系与弱活力自交系杂交后代分析功能标记的选择效应。本研究的完成不仅加深对甜玉米种子老化调控机制的认识，且将为甜玉米种子耐老化遗传改良提供技术支撑。

甜玉米因胚乳淀粉合成基因突变抑制淀粉合成，胚乳皱缩，粒重轻，极不易贮藏。因此种子耐老化基因的克隆及分子机制解析是培育耐老化甜玉米品种的首要条件。本研究中利用高代回交系群体、RIL群体和甜玉米自交系关联群体对甜玉米种子耐老化基因进行定位研究，结合亲本的转录组分析进行了候选基因分析，选择RIL群体中的极端株系和不耐老化亲本杂交自交得 F2:3家系群体，对耐老化候选基因进行了精细定位研究，进一步加密标记筛选到3个分子标记与种子耐老化表型共分离，在育种群体进行标记的功能验证，将候选区间锁定在18个基因范围内。筛选出候选基因Zm00001d015090进行了超表达和基因突变，初步研究了其参加甜玉米种子耐老化的功能，超表达材料人工加速老化后，发芽势和发芽率与亲本差异不显著，但种子活力指数显著高于亲本，突变体材料人工加速老化后，种子活力指数显著低于亲本，亚细胞定位显示该基因定位于细胞核。