苹果花粉特异MAPK在花柱S-RNase作用下参与自交不亲和反应研究

Based on the previous study, apple self-incompatibility is apple style S-RNase leads to accumulation of pyrophosphoric acid, and eventually causes pollen tube inhibition in self-pollinated reaction. Meanwhile, we used protein high-throughput sequencing to find the differential expression proteins between the style of self and cross pollination, and obtained a pollen specific MAPK protein. However, the mechanism of self S-RNase contribute to pollen tube inhibition is still unclear. In our study, we firstly explore the MAPK signal transduction pathways activated by S-RNase in the pollen tube; secondly, using antisense oligonucleotide chain culture pollen tube to silence MAPK gene and then added the S-RNase protein, we will observe the pyrophosphate accumulation and pollen tube growth, to clear if it is ture that S-RNase could inhibite pollen tube growth and pyrophosphate accumulation by regulating MAPK; again, fingding the interaction protein of MAPK which might be phosphorylated by MAPK, this is to clear that if the interaction protein caused the accumulation of pyrophosphate in pollen tube; at last,we will confirm that MAPK participate in the self-incompatibility by genetic transformation. Above all, we could clarify that MAPK play a role in self-incompatibility reaction under the induction of S-RNase.

前期研究发现，苹果的自交不亲和性是自花授粉的花柱S-RNase在进入花粉管后会引起焦磷酸大量积累，阻碍tRNA氨酰化，阻止花粉管生长；同时，自花与异花授粉后蛋白质高通量测序比较分析寻找到一个花粉特异表达且受自我S-RNase诱导的MAPK家族蛋白；但MAPK蛋白是如何在自我S-RNase作用下参与焦磷酸积累并引起花粉管停止生长的机制尚不清楚。鉴于此，本研究首先明确S-RNase进入花粉管所激活的MAPK信号转导路径；其次，反义寡核苷链转染沉默培养中花粉的MAPK基因再添加S-RNase蛋白，明确S-RNase是否通过MAPK调控焦磷酸积累抑制花粉管生长；再次，寻找受MAPK磷酸化的互作蛋白，明确S-RNase是否通过MAPK磷酸化其下游蛋白而引起了花粉管焦磷酸的积累；最后验证MAPK其是否参与了自交不亲和反应。最终明确苹果花粉特异MAPK在花柱S-RNase作用下参与自交不亲和反应的机制。

苹果是典型的配子体自交不亲和植物，自花授粉时花粉管会发生自交不亲和反应，导致授粉受精失败，进而无法坐果。本项目以苹果为试材，解析了以MAPK基因为核心的苹果自交不亲和反应下花粉管信号通路，经自我、异我 S-RNase 处理、自交和异交比较分析，鉴定和挖掘出MAPK、OXI、PTI激酶类等自交不亲和反应关键因子，均参与自我 S-RNase 介导的 ROS 信号通路，MAPK、OXI、PTI等激酶通过磷酸化反应传递自交不亲和信号的分子机制。同时解析了亚精胺调控苹果自花结实性的分子基础，明确了亚精胺与MAPK等激酶介导的信号通路之间的关联性，构建出自交不亲和反应花粉管伸长停滞信号传递网络。此外，在本项目支撑下，以自花结实特异资源为材料，发掘自花结实关键变异2个，并基于此开发出可用于自花结实育种分子辅选标记。项目成果发表研究论文4篇，获批国家发明专利4项，获神农中华农业科技奖一等奖1项，培养博士3名。