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。

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.