水稻NP4基因控制光（温）敏雄性不育分子机理

Hybrid rice, which successful used the male sterility, led to a great improvement in rice productivity and met the food demand of the growing population in the past decades. A rice incomplete dominant male-sterile mutant showed slightly dwarf, sheathed panicle, slender filament and no mature pollen grains was identified from the T-DNA insertion mutant library. From the images of semithin section, the mutant’s tapetum showed looser structure than wild type at microsporocyte formation stage and failed to form pollens subsequently. Using an F2 population from a cross between the mutant and the indica rice cultivar Longtefu B, the mutant gene locus was delimited to a 103 kb region on chromosome 4. It was also detected that the mutant phenotype was co-segregated with the T-DNA insertion. By isolated flanking sequence, the insertion was found occurred in intergenic regions on chromosome 4 and located at the previous 103kb mapping region. The expressions of two upstream and two downstream genes of T-DNA inserted site respectively were detected. And the expression level of the first downstream gene，which encoded an AT-hook transcriptional factor, was significantly increased at every stage in the process of microspore development. We designated the mutant gene as No Pollen 4(NP4). Then, the candidate gene was confirmed by overexpressing NP4 derived by Actin1 promoter in wild type. The anther and pollen grain morphologies of overexpressing NP4 plants were similar with np4. At the later, we found that when the initiation of panicle development occurred in late September to early October (low temperature and SD), the morphologies of anthers and pollen grains in np4 were mostly normal. Million while, when the rice panicle development initiated in early March(SD and relative low temperature) in Sanya,, np4 exhibited close-to-normal anther and pollen grain morphologies, which suggesting that the male sterility in np4 may be controlled by photoperiod or temperature. Interestingly, the expression level of PMS3 gene, which controlling genic male sterility in NK58S and PA64S, was decreased dramatically in np4 compared with the wild type, while the expression level of other detected photoperiod-sensitive genic male sterile (PGMS) and thermo-sensitive genic male sterile (TGMS) genes showed no obvious changed. When overexpress the PMS3 gene in np4, the transformed plants exhibited close-to-normal anther and pollen grain morphologies. This research will aim to identify the type of photoperiod-sensitive or thermos-sensitive in np4, discover the signal pathway of NP4 gene involved in controlling PGMS or TGMS, and demonstrate the genetic relationship between NP4 and PMS3 as well. Further study on biological functions of NP4 will enrich our understanding of the molecular basis of photoperiod- and thermo-sensitive genic male sterility, and finally help for rice heterosis utilization and molecular breeding design.

雄性不育的成功利用使水稻产量取得了极大提高，为确保我国粮食安全起到了极为重要的作用。从水稻T-DNA插入突变体库中筛选到一个不完全显性雄性不育突变体，利用图位克隆结合T-DNA标签法确定位于第4染色体上1个编码AT-HOOK转录因子的基因为候选基因，命名为No Pollen 4(NP4)，转基因过量表达NP4可以重现np4的突变表型。随后发现np4为一光（温）敏突变体，对几个已知光（温）敏基因进行表达分析，发现其中PMS3基因（来源于NK58S和PA64S）表达量在np4突变体中显著下降，利用转基因实验在np4中过量表达PMS3基因，np4表型恢复为野生型。拟对np4进行系统的光温鉴定，明确NP4参与光温敏不育的信号途径，确定NP4基因与PMS3基因的遗传互作关系进行研究，阐明NP4调控水稻光（温）敏核不育的分子机理，为水稻杂种优利用分子设计育种提供新的基因资源。

从水稻T-DNA插入突变体库中筛选到一个显性雄性不育突变体，对np4进行了深入的表型鉴定和基因克隆，发现在温度32℃的条件下，np4无花粉形成，败育彻底，而在24℃条件下，np4育性完全恢复，表明np4为一温敏核不育突变。对花粉发育的各个时期进行了树脂切片观察，发现np4在小孢子母细胞形成阶段，绒毡层发育出现异常，导致最终无花粉粒形成。随后克隆了NP4基因，NP4编码AT-HOOK家族转录因子,NP4具有转录激活功能。通过RT-PCR分析发现，光温敏基因PMS3在np4突变体中表达量明显降低，提示NP4可能通过PMS3调控花粉育性，通过ChIP实验，验证了NP4蛋白可以结合PMS3启动子区域，构建PMS3过量表达载体转化np4突变体，np4的不育表型可以恢复，进一步表明np4通过调控PMS3起作用。筛选了NP4的自然变异，发现NP4存在明显的籼粳分化，提示NP4在自然选择和驯化中发挥了重要作用。本研究为NP4基因在水稻两系杂交稻制种中的进一步利用提供了基础。