水稻粒长基因GL3.3功能解析

Grain size is an appearance quality trait correlated to grain yield in rice. Cloning of grain size gene is meaningful for rice quality improvement. 529 accessions of a worldwide core collection were used for genome-wide association mapping for grain length. A candidate gene, GL3.3, underlying the novel association locus on chromosome 3, was defined. GL3.3 encodes a transcription factor. Both T-DNA insertion mutant and genetic complementation test have validated its identity. GL3.3 has a higher expression level both in younger panicle and endosperm, and regulates the expression of the cell cycle related genes. Here, we will further examine the temporal and spatial expression pattern of GL3.3. Its encoding protein will be subcellular localized and be examined for the activity of transcription. Both RNA-Seq and ChIP-Seq assays will be used to identify the down-stream genes regulated by GL3.3. The target genes direct binding GL3.3 will be validated by yeast one-hybrid assay and electrophoretic mobility shift assay. In addition, the interaction protein with GL3.3 will be identified using both yeast two-hybrid assay and Co-IP assay. Thus, it would be clear how GL3.3 regulates grain length. Moreover, re-sequencing of GL3.3 in wild rice and cultivars will be performed to reveal the favorable allele. This project will elucidate the molecular mechanism of GL3.3 controlling grain length and provide favorable alleles for improvement of grain appearance quality.

水稻粒形既是产量性状又是外观品质性状。克隆粒形基因对水稻产量与品质改良具有重要意义。前期我们利用529份世界水稻核心种质群体，对粒长进行了全基因组关联分析，在第3染色体上鉴定到一个粒长候选基因GL3.3，该基因编码一个转录因子。经突变体与遗传互补证实该基因调控粒长。GL3.3在幼穗与胚乳中表达较高，它调控细胞周期基因的表达。本研究将开展GL3.3的组织表达特异性、蛋白质亚细胞定位与转录激活或抑制活性分析。进而，结合RNA-Seq与ChIP-Seq技术检测GL3.3的下游调控基因，并利用酵母单杂交与凝胶电泳迁移率分析鉴定它的直接下游靶基因。同时，利用酵母双杂交与Co-IP技术筛选与验证它的互作蛋白。进而明晰GL3.3调控粒长的分子机理。此外，重测序自然群体与野生稻的GL3.3，鉴定出优异等位基因。从而为水稻产量与外观品质改良提供理论指导和基因资源。

粒长既是稻米外观品质性状，也是产量性状。鉴定与克隆粒长新基因不仅有助于探究粒形变异调控机理，而且可用于育种改良稻米产量与外观品质。本研究克隆了粒长基因GL3.3，并通过遗传转化进行了功能验证。研究发现GL3.3在水稻多个组织中均有表达，在幼穗2～4 cm时期表达水平最高；GL3.3蛋白定位于细胞核内，但不具有转录激活活性；GL3.3可能通过调控细胞大小改变水稻粒长，且生长素合成基因很可能参与其中的调控；鉴定其互作蛋白或DNA将有助于进一步阐释该蛋白的功能及其调控机理。通过对水稻种质资源材料中的GL3.3不同等位基因分析得出单倍型hap6是有育种应用价值的优异单倍型。此外，本研究还克隆了其同源基因LOC_Os07g42590，且完成了功能验证。研究表明该同源基因参与调控粒宽，且发现它与GL3.3不存在功能冗余。因此，通过应用GL3.3和LOC_Os07g42590的优异单倍型可改良稻米粒形，进而实现对其产量及外观品质的定向改良。