水稻种子萌发和愈伤分化基因GR9的遗传定位和功能研究

Both of seed germination and callus differentiation are starting processes of plant organ initiation. Although it has been shown that they are regulated by different plant hormones, no one can rule out the possibility that common regulator(s) may exist. Previously, we constructed a set of chromosome segment substitution lines with indica 9311 as donor and japonica Nipponbare as receptor, respectively. With that, a major locus qGR9 that regulates seed germination was detected on chromosome 9 in rice genome. Transgenic study indicated that a transcription factor, locating in the region of qGR9, might be a good candidate gene which was named as GR9 in our study. T-DNA line of GR9 (gr9) showed pre-harvest sprouting phenotype while the overexpressors displayed late germination phenotype. Meanwhile, transgenic study showed that GR9 might also play an important role in differentiation of callus. It was found that callus differentiation rate was greatly reduced in GR9 overexpressors, while gr9 mutants showed opposite phenotype. gr9 mutants expressing indica GR9 from 9311 also showed significant lower callus differentiation rate compared with that expressing japonica GR9 from Nipponbare, and a single nucleotide polymorphism in the coding region might be the cause of this difference in gene function. Further, we will study the molecular mechanisms that GR9, as a common regulator, regulates both seed germination and callus differentiation in rice. Through this work, a key gene that control seed germination and callus differentiation will be discovered and new strategies to solve the problems of rice pre-harvest sprouting and low transformation efficiency in indica varieties will be provided.

种子的萌发和愈伤的分化都是控制植物器官分化的起始过程。过去研究显示二者可能受不同的激素控制，但不排除受共同的因子调控。本研究前期利用一套以9311为供体，日本晴为受体的替换系，分离到一个影响种子萌发的主效位点qGR9，并获得一个可能的候选基因GR9。研究发现gr9突变体种子更易穗上发芽，而过表达GR9的种子萌发速率明显减慢。另一方面，过表达GR9的水稻愈伤分化效率急剧下降，而突变体gr9的愈伤分化效率明显提高。将9311和日本晴GR9分别转化至gr9突变体中发现籼稻GR9转化系愈伤分化效率显著低于粳稻GR9转化系，而导致籼粳GR9发生功能差异的关键变异可能是位于该基因外显子中的一个SNP。本项目将对GR9在水稻种子萌发和愈伤分化过程中的生物学特性和功能进行深入研究，以期揭示这两个生理途径间调控的关联性，发掘影响籼稻愈伤分化效率的关键基因，为解决穗发芽和籼稻遗传转化效率低下问题提供方案。

籼粳间的愈伤分化效率存在明显差异，不利于籼稻的遗传转化，但是人们对其中的遗传机制却知之甚少。本研究通过遗传分析在水稻基因组中鉴定获得一个可以同时影响水稻种子萌发和愈伤分化效率的主效QTL位点qGR9。遗传定位、转基因功能验证和亚细胞定位结果表明GR9是一个影响水稻愈伤分化效率的负调控因子，主要在水稻细胞核内行使功能。gr9突变体的愈伤分化效率显著上升；而GR9过表达后，水稻愈伤分化效率急剧下降。将籼型GR9转入粳稻日本晴后，水稻愈伤分化效率显著下降；而将粳型GR9转入替换系N36（含有籼型GR9）中后，水稻愈伤分化效率获得改善。同时，研究结果表明籼粳间GR9编码区的1个SNP对水稻愈伤分化效率影响较大。将粳稻GR9该SNP突变成籼型后，转基因水稻愈伤的分化效率显著下降，与籼型GR9转基因愈伤的分化效率较为相似，由此表明籼型GR9可能是导致籼稻愈伤分化效率较低的重要基因之一。酵母双杂交、BiFC和CoIP试验结果表明，GR9可以与细胞分裂素信号传递途径因子OsAHP2和生长素信号传递途径因子OsADA2相互作用，且OsAHP2或OsADA2缺失后，水稻再生效率显著下降，由此表明GR9可能同时通过细胞分裂素和生长素信号传递途径影响水稻再生效率。综上，本研究综合利用遗传学、分子生物学和生物化学方法在水稻基因组中成功分离获得影响水稻种子萌发和愈伤分化的主效基因GR9，并对GR9影响愈伤分化的分子机制进行了较为深入的研究，初步建立了GR9的作用模型，该研究结果可为未来籼稻高效遗传转化平台的建立提供新的思路和方法。