苹果砧木平邑甜茶PcG蛋白MhMSI1调控单性生殖的分子机理

Parthenogenesis is an essential element in apomictic process, which has a huge potential value in agriculture. Recent studies showed that it might be regulated by DNA methylataion, however, it is yet unclear about its regulatory molecular mechanism in plants. In our previous study, we found that the expression level of MhMSI1 was highly correlated with the capacity of parthenogenesis and the transcriptional levels of parthenogenesis-related genes. Meanwhile, MhMSI1 also negatively regulated the expression level of methyltransferase gene MhMET1, suggesting that MhMSI1 maybe regulate parthenogenesis via DNA methylation. In this study, Pull-Down and BiFC assays will be used to verify the interaction between MhMSI1 and MhRBR1, ChIP-PCR and EMSA assays will be performed to check if these complex regulate MhMET1. Subsequently, DNA methylation and expression levels of parthenogenesis-related genes will be detected in a separate population of tea crabapple and MhMSI1 overexpression transgenic apples, respectively, followed by an analysis about the relationship between MhMSI1 and parthenogenesis. Finally, parthenogenetic capacity in MhMSI1 transgenic apples will be analyzed. This study aims to elucidate the molecular mechanism of parthenogenesis modulated by PcG protein MhMSI1. The results might provide theoretical basis for the obtaining of apomictic resources by genetic modification in apple.

单性生殖作为无融合生殖的关键组分，在生产中有巨大的应用潜力。研究表明单性生殖受DNA甲基化调控，但具体分子机制尚不明确。前期研究发现，MhMSI1表达水平与单性生殖率及单性生殖相关基因的转录水平高度相关；同时，MhMSI1负调控甲基转移酶基因MhMET1的表达，表明MhMSI1可能通过DNA甲基化调控单性生殖。本研究拟采用Pull-Down和BiFC技术验证MhMSI1与MhRBR1的相互作用，通过EMSA检测该复合体对MhMET1启动子的结合，并用瞬时表达系统检测MhMET1对单性生殖相关基因DNA甲基化的影响；随后拟在单性生殖分离群体中和前期获得的过量表达MhMSI1转基因苹果中检测单性生殖相关基因的DNA甲基化水平和表达水平；最后，统计MhMSI1过量表达转基因苹果的单性生殖率，进而揭示MhMSI1调控单性生殖的分子机理，为利用遗传改良获得无融合生殖特性的苹果属资源提供理论依据。

无融合生殖现象在蔷薇科苹果属果树中较为常见，但对多年生木本果树的无融合生殖分子机理知之甚少。苹果砧木平邑甜茶的无融合生殖由单性生殖、无融合非减数分裂和胚乳的自发发育构成，为研究无融合生殖的分子机理，本研究利用平邑甜茶与有性生殖的苹果舞美进行了杂交，根据后代的遗传分离规律将平邑甜茶的无融合生殖特性分为无融合非减数分裂和单性生殖两个独立遗传的发育事件，并在杂交后代中分别获得这两个事件的分离群体，通过高通量测序，针对单性生殖性状进行了表达谱分析，克隆了一些潜在的无融合生殖相关基因MhMSI1，通过功能验证发现转基因苹果具有一定的单性生殖能力，种子较小。酵母双杂与Pull-Down实验表明MhMSI1与MhRBR1相互作用，并且可以结合在MhMET1启动子上，MhMET1在转基因苹果中表达下调，转基因苹果的全基因组的DNA甲基化水平明显降低；且影响种子发育的SERK1基因表达上调，而甲基化水平下调。除此之外，MhMSI1异源转化番茄影响果实的成熟，提高了番茄的单性结实率。项目研究成果将为苹果属无融合生殖分子调控机理问题提供重要信息，推动无融合生殖理论发展及其在农业生产中的应用。