一个无花粉型水稻雄性不育新基因的图位克隆及其分子机制研究

Researches on process of male reproductive development in rice and related molecular mechanisms are particularly important since rice acts as not only an important food crop, but also a model plant of monocots. Studies on the regulators and molecular mechanism of the rice reproductive development by using rice male sterile mutant has been always a focal point for rice bioresearch. In earlier stage of this research, we identified a no-pollen male sterile mutant which exhibited snow-white anthers without mature pollen grains and completely sterile spikelets, that were significantly abnormal compared to the wild-type. Genetic analysis revealed that the mutation trait was controlled by a single recessive genic gene, which was fine-mapped to a 41-kb region on the long arm of chromosome 4 between markers 4MD-10 and 4MD-11. Gene prediction analysis indicated that no rice male sterility allele has been reported in this positional interval. Therefore, we believe that it is a novel gene dominating rice male sterility. Seqence analysis reveals that the mutant carries a single nucleotide substitution which leads a substitution of one amino acid in the third exon of ORF2. Thus, this gene was recognized as the candidate gene. This project intends to obtain the target gene through map-based cloning and genetic transformation methods, and systematically analyze its expression patterns, biological functions, and interaction regulatory networks, etc., which will contribute to the comprehension of its molecular mechanism regulating male reproductive development in rice.

水稻作为重要的粮食作物，又是单子叶植物的模式植物，其雄性生殖发育进程及其相关分子机理研究尤为重要。利用雄性不育突变体研究水稻雄性生殖发育的相关基因及其分子机理，一直都是水稻生物学研究的重点和热点。本项目前期分离鉴定出一个无花粉型水稻雄性不育突变体，该突变体花药雪白、无花粉、小穗完全不育，较野生型差异显著。遗传分析表明，该突变性状受一对隐性核基因控制；现已将该雄性不育基因精细定位至水稻第4号染色体长臂介于分子标记4MD-10和4MD-11之间约41-kb范围内，该区间内未见已报道的雄性不育基因，是一个新的水稻雄性不育基因。测序结果显示，突变体在其中ORF2的第三个外显子区发生了一个单碱基替换，导致其编码的氨基酸发生了改变，故将该基因确定为候选基因。本项目拟通过图位克隆和遗传转化方法获得该目的基因，并对其表达模式、生物学功能和互作调控网络等进行系统分析，以期探明其调控水稻雄性生殖发育的分子机理。

雄性不育相关调控因子的挖掘及其作用机理研究，对于解析水稻生殖发育的分子调控网络和杂种优势利用及品种改良，具有重要的双重指导意义。本研究以无花粉型雄性不育突变体bm1为研究材料，鉴定了bm1的花粉育性和小穗育性，细胞学观察证明突变体bm1的绒毡层发育与降解异常，不能合成乌氏体，使得小孢子后期发育营养供给不足，最终降解，导致无花粉表型。脂肪酸指标测定结果证实其花药角质层和花粉外壁前体可能没有正常合成或分布。图位克隆实验结果表明R2R3类MYB转录因子基因BM1上的单碱基替换（A→T）是导致突变体bm1雄性不育表型的原因。TUNEL检测、DAPI染色和表达分析结果表明，BM1反向调控绒毡层的降解，对减数分裂有较小影响，但并不调控小孢子生成过程中的胼胝质合成与降解。自激活实验和蛋白互作实验证明，BM1与TIP2、EAT1和TIP3及其本身互作。双突变体表型和qPCR分析结果证实BM1作用于CYP703A3和CYP704B2上游。本项目所得结果克隆了水稻无花粉型雄性不育基因BM1并明确了其调控水稻雄性生殖发育的分子机制，也为智能不育系创制和新兴杂种优势利用途径提供了理论指导和基因资源。