DEAD-BOX类RNA解旋酶AtFGA1调控拟南芥花粉管导向和配子识别的分子机理研究

Fertilization is essential for the production of seed and fruit in plants, and is required for the completion of life cycle and reproduction. Despite of its vital importance, the molecular mechanisms underlying fertilization in plants remains largely unknown. Guidance of pollen tube by the ovule and the gamete recognition are necessary processes of fertilization. Previously, we identified a mutant of DEAD-BOX RNA helicase in Arabidopsis and annotated it as atfga1-1/+. In this mutant, gametophyte development of both sexes was normal, but the seed production and female gametophyte transmission efficiency were reduced. Further analysis revealed that defect in AtFGA1 influenced pollen tube guidance that controlled by the ovule as well as the gamete recognition process. In this project, we intend to further analyze the phenotype of atfga1-1/+, detect the expression pattern of AtFGA1 and its encoding gene within the embryo sac, investigate the enzyme activity of AtFGA1, study the function of non-conserved domain at both N and C terminus of this protein, and identify proteins and mRNAs interacting with AtFGA1. We expect that the results of this project can shed new light on the molecular mechanisms of plant fertilization.

植物受精过程是产生种子和果实的前提，也是植物完成生活周期和繁殖后代的必经阶段，然而植物受精的分子机理尚不清楚。胚珠引导花粉管的定向生长和配子识别是植物受精过程的重要步骤。我们在前期工作中鉴定出一个拟南芥DEAD-box类RNA解旋酶编码基因的突变体atfga1-1/+。该突变体雌雄配子体发育正常，但结实率下降、雌配子传递效率降低。初步分析发现AtFGA1基因突变造成胚珠不能正常吸引花粉管，雌雄配子识别出现异常。本项目拟在前期工作的基础上，通过进一步分析atfga1-1/+突变体表型、明确AtFGA1基因及其编码蛋白在胚囊中的表达模式、分析AtFGA1突变对配子融合的影响、检测AtFGA1的解旋酶活性及其N端和C端非保守区域功能、分离与AtFGA1结合的mRNA、鉴定AtFGA1的互作蛋白，研究AtFGA1在调控拟南芥花粉管导向和配子体识别过程中的作用机制，为理解植物受精的分子机理提供新信息

AtFGA1在胚囊发育过程中以及胚和胚乳中均有表达信号。N端50个氨基酸和第433-522位氨基酸决定了AtFGA1的核定位，而第310-422位氨基酸决定其核仁定位。受精过程中约有13%的花粉管在atfga1-1/+珠孔附近缠绕生长，但这些花粉管最终都能够进入珠孔。pHTR10::HTR10-mRFP标记显示，atfga1-1/+ 雌配子体能够正常完成受精。进一步的表型分析发现，atfga1-1/+ 雌配子受精后有25%左右的胚停留在球形胚阶段，导致种子败育。GPR23::GUS标记结果显示，当atfga1-1/+ 发育到球形胚时期时，胚乳退化。利用AtFGA1对酵母突变株进行互补实验，发现其能互补SPB4和HAS1突变株的热敏感表型。对pre-rRNA和印记基因进行定量和半定量RT-PCR检测结果表明，AtFGA1参与拟南芥pre-rRNA的加工，同时参与了对印记基因pre-mRNA的剪接。推测AtFGA1通过对pre-rRNA和印记基因pre-mRNA的剪接参与了胚和胚乳的发育过程。研究结果对于理解RNA剪接介导的胚和胚乳发育的分子机理提供了新的重要信息。