苹果花粉γ-硫堇蛋白在自交不亲和反应中的作用机制研究

S-RNase is a determinant factor of self-incompatibility in apple style. Using yeast two hybrid assay, a γ-thionin gene was obtained by screening a pollen cDNA library with S- RNase as bait. Pre-experiment of pollen culture showed that γ-thionin may take part in self incompatibility by acting as an inhibitor of S-RNase activity in apple pollen. In this proposal, we will first make point mutation on S-RNase to test whether γ-thionin can interact with the activity domain of S-RNase. In order to clarify whether γ-thionin can inhibit the activity of S-RNase, we will incubate γ-thionin and S-RNase proteins to culture the apple pollen and then test the activity of S-RNase; antisense oligonucleotides transfection assay will then be employed to silence γ-thionin gene in apple pollen and petunia will be used to silence its homologue, and the resulting apple and transgenic petunia pollen will be used for self and cross pollination to test the pollen growth and seed setting rate. Finally, We will use fluorescence in situ hybridization to mark the location of γ-thionin and S-RNase proteins in pollen tube after self and cross pollination to identify the interaction between γ-thionin and S-RNase and the way that γ-thionin inhibits the activity of S-RNase. .The aim of this proposal is to prove whether γ-thionin participate the self incompatibility process and whether the participation is occurred through inhibiting S-RNase activity. The predicted result will provide new thought for studies on plant self incompatibility.

S-RNase是苹果花柱自交不亲和决定子。我们用S-RNase为诱饵筛选花粉酵母cDNA文库获得一个γ-硫堇，从γ-硫堇削弱S-RNase抑制花粉管生长等离体实验初步推断γ-硫堇可能作为S-RNase活性抑制剂参与了自交不亲和反应。因此，本研究首先通过S-RNase活性位点及其突变前后与γ-硫堇体内外互作明确γ-硫堇是否只结合到S-RNase活性位点区；其次通过两蛋白共孵育检测S-RNase酶活及添加培养花粉管明确γ-硫堇是否能抑制S-RNase活性；最后利用反义寡核苷酸转染沉默γ-硫堇的苹果花粉及转矮牵牛γ-硫堇的花粉分别自异花授粉，观察花粉管生长，统计结实率，并通过双荧光原位免疫杂交技术标记两种蛋白在苹果自异花授粉后花粉管内位置，判定γ-硫堇能否调控自交不亲和反应，观察γ-硫堇是否与S-RNase结合及可能抑制其活性方式。最终明确γ-硫堇通过抑制S-RNase活性参与自交不亲和反应。

本课题立项于2013年，计划开展苹果花粉γ-硫堇蛋白在自交不亲和反应中的作用机制研究。本研究以苹果为试材，经过2014-2017年四年的努力，利用多种技术手段证明了S-RNase在进入花粉管过程中引起了Ca2+-ROS-JA-MdMYC2的一系列信号反应，诱导了花粉管中一个具有γ-硫堇结构域的防御蛋白的响应，防御应答S-RNase进入花粉管，且抑制S-RNase活性。其主要研究结果如下：1. 我们从钙信号研究入手，验证了S-RNase可以打破花粉管内钙离子梯度，影响钙响应蛋白MdCBL5表达，使得花粉管尖端ROS信号减弱，亚顶端显著增强，诱导ROS相关基因MdRbohH上调表达，JA明显升高，MdMYC2转录水平上调表达。表明S-RNase可诱导Ca2+-ROS-JA-MdMYC2信号反应。2. 重组MdMYC2蛋白Chip-seq苹果基因组，获得了γ-硫堇蛋白基因MdD1的启动子序列，酵母单杂交验证了MdMYC2与MdD1启动子序列的结合，并证明MdD1的表达受MdMYC2转录调节，表明MdD1受Ca2+-ROS-JA-MdMYC2信号路径的激活。免疫荧光定位结果发现：添加S-RNase后，随着时间的延长， MdD1向花粉管顶端和亚顶端膜方向移动，自我和异我花粉管的MdD1均具有响应S-RNase入侵的作用；而沉默MdMYC2，花粉管中MdD1则并未发生位移，说明MdD1通过MdMYC2响应S-RNase的入侵。3. 进一步研究发现MdD1可以抑制S-RNase活性的发挥。MdD1与S-RNase的活性位点区域互作，与非活性位点区域不互作。该研究结果进一步完善了苹果自交不亲和性分子机制，为苹果花粉与花柱识别和自花授粉后花粉管致死的研究奠定基础。

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