休眠基因RDO5的抑制因子SUR1和SUR2调控种子休眠的分子机制

Seed dormancy plays an adaptive role in nature by optimizing germination at the most suitable time. A tight control of dormancy is important in many crop species. Low seed dormancy is required for uniform and fast germination after sowing, and further growth potential; On the other hand, Low levels of dormancy can cause pre-harvest spouting, which can cause economic losses to cereal production including reductions in seed quantity and quality. RDO5 is a newly cloned seed dormancy gene. RDO5 affects seed dormancy without any pleiotropic phenotypes. And RDO5 regulate seed dormancy in an ABA-independent way. Previous research indicates RDO5 may involve in seed stored mRNA regulation and stored protein modification to determine seed germination or not. But the molecular mechanism is still unknown. In order to uncover the mechanism that RDO5 involved, in this project we did EMS mutagenesis screen with rdo5-2, the mutant of RDO5 with very low dormancy. And we got 2 mutants that can rescue dormancy phenotype of rdo5-2, named sur1 (suppressor of RDO5-1) and sur2. We will clone SUR1 and SUR2 through genomic DNA sequencing and SNP linkage analysis. Then we will investigate the detailed mechanism of dormancy regulation by SUR1 and SUR2. We will study their functions, such as gene expression pattern, interaction proteins, and downstream genes on genetics, molecular biology, proteomics and bioinformatics levels and approaches. We also will investigate the crosstalk or difference between RDO5, SUR1, and SUR2. These investigations will further deepen the mechanism of seed dormancy regulation and build up RDO5 seed dormancy regulation pathway or network.

种子休眠确保种子在最适宜的条件下萌发，对植物体的生存、延续和进化起着积极的作用。种子休眠的强弱关系到种子穗发芽、萌发率、整齐度及后续生长，对作物的产量和品质十分重要。RDO5是一个最新克隆、特异影响种子休眠的基因，通过不依赖ABA途径特异影响种子休眠。前期研究发现RDO5可能通过对种子中存储mRNA的调节和存储蛋白修饰来调节种子休眠，但是其具体途径和分子机制还很不清楚。本项目计划EMS诱变完全丧失休眠的突变体rdo5-2，获得休眠表型完全或部分恢复的突变体。然后通过全基因DNA测序和SNP关联分析克隆rdo5-2抑制因子、调节种子休眠的新基因SUR1和SUR2。然后从SUR1和SUR的基因表达模式、互作蛋白、上下游基因等方面，对其调节种子休眠分子机制展开研究，并分析SUR1、SUR2 与RDO5调控途径的交叉互作。本研究将加深对种子休眠及RDO5调控途径和网络的认知。

合适的种子休眠程度确保了植物物种的存活与进化，进而决定着人类社会的农业生产发展。鉴于种子休眠具有如此重要的生物学和农业生产上的意义，因此深入理解种子休眠形成与解除的生物学机理将是具有重大现实价值的。在本研究中，我们鉴定得到了新的与拟南芥种子休眠相关的基因ODR1和ODR2。表型分析结果表明：ODR1负调控种子的休眠并可降低成熟种子中的脱落酸 (ABA) 含量。ODR1蛋白定位于细胞核中且特异在种子中表达，其在种子中的具体表达模式为：ODR1的表达水平随着种子的逐渐成熟而不断升高，成熟种子经历吸胀后，ODR1的表达水平迅速降低。酵母单杂交，染色质免疫共沉淀以及双荧光素酶瞬时表达实验结果表明：ABI3可结合至ODR1启动子上的RY顺式作用元件，负调控ODR1基因的表达。结合酵母双杂交文库筛选，酵母双杂交与双分子荧光互补以及免疫共沉淀实验验证，我们获得了ODR1的一个互作核蛋白bHLH57。酵母单杂交，染色质免疫共沉淀以及双荧光素酶瞬时表达实验结果表明：bHLH57可结合至NCED6与NCED9启动子上的E-box顺式作用元件，正调控NCED6与NCED9基因的表达。进一步地，通过双荧光素酶瞬时表达实验以及双突变体odr1-2 bhlh57-2种子中基因表达水平检测，我们发现：ODR1可以通过结合bHLH57，进而抑制bHLH57正调控NCED6与NCED9基因表达的功能，从而降低种子中的ABA含量。综上所述，我们阐明了在拟南芥种子中存在的由ABA，ABI3，ODR1，bHLH57与NCED6和NCED9共同参与的种子休眠调控通路。