肌醇1,4,5-三磷酸（IP₃）是动植物体内一种非常重要的第二信使，由4，5-二磷酸磷脂酰肌醇（PIP2）水解产生。大量研究表明，动植物体内存在IP₃–Ca^2＋信号途径，IP₃能够引起胞内钙离子增加。动物细胞中IP₃受体（IP₃R）的作用机制已经比较清楚，但在植物中目前为止未曾找到具有明确结构和功能的IP₃下游蛋白。本实验室前期利用GSK细胞（敲除STIM1/2，并且转染GCaMP6钙离子指示剂的人胚胎肾细胞），从拟南芥cDNA文库中筛选到了一个利用毒胡萝卜素（TG）清空内质网钙库后，能够介导胞外钙离子内流引起胞内钙离子增加的cDNA片段。通过序列比对，发现其为具有磷脂酸（PA）结合特性的蛋白—PLD regulated protein 1（PLDrp1）的C端片段。进一步的研究表明，该蛋白能够特异结合生理浓度范围的IP₃，并且与Annexin 4（ANN4）共同介导Flg22引起的胞内钙增加及植物的抗病反应。因此，将其重新命名为IP₃感受器（IP₃S）。

本论文主要研究IP₃S结合IP₃的结构与功能关系，对其结构区域进行功能划分，形成IP₃S的初步结构功能域拓扑图。首先，根据IP₃S蛋白的一级结构及三维结构预测图，发现该蛋白为一种没有完整结构域和三维结构的无序蛋白；在其N端110位氨基酸处开始出现14段由12-15个氨基酸组成的保守重复序列，以及从第90位氨基酸处开始不再出现预测的短a-螺旋结构。据此，选择90位氨基酸处将IP₃S进行截断，构建出IP₃S的N端（1-90位氨基酸，IP₃S-Nt）及C端（91-381位氨基酸，IP₃S-Ct）两部分截断片段的原核表达载体。其次，利用大肠杆菌菌株Transeeta表达并纯化了IP₃S-Nt及IP₃S-Ct片段，通过微量热涌动（MST）实验，检测到IP₃S-Nt不结合IP₃，而IP₃S-Ct结合IP₃，且结合Kd值与IP₃S全长无差异。进一步采用同样策略，对IP₃S的蛋白片段IBD-1（90-143位氨基酸残基）及IBD-2（144-181位氨基酸残基）进行原核表达及纯化。MST实验结果表明，IBD-2结合IP₃，但结合Kd值大于全长IP₃S及其C端，而IBD-1不结合IP₃。

另外，利用荧光素酶互补实验方法验证了IP₃S与ANN4的互作区域。首先，构建了IP₃S-Nt及IP₃S-Ct的cLUC载体，与ANN4-nLUC共转染烟草叶片后发现烟草叶片有强烈的自发荧光，即IP₃S的N端及C端都与ANN4产生互作。其次，采用同样的策略验证了IP₃S片段T1-180（1-180位氨基酸残基）及T181-381（181-381位氨基酸残基）与ANN4均存在互作。这样的结果暗示着IP₃S似乎并不存在与ANN4互作的特定区域，而IP₃S是否与ANN4的不同区域产生或作，这需要进一步的实验验证。

最后，本论文根据IP₃S特异结合IP₃的特点，仿照钙离子指示剂YC3.6以及其优化型TurCaMP7钙离子指示剂，在IP₃S的N端及C端分别连上供体蛋白mTurquoise2（蓝色荧光蛋白CFP的优化突变体）及受体蛋白mNeonGreen（绿色荧光蛋白GFP突变体变型），构建了IP₃的指示剂。根据荧光共振能量转移（FRET）的原理，验证IP₃S结合IP₃后是否会产生构型变化。研究结果表明，IP₃S结合IP₃后，指示剂并没有显著的FRET变化。这符合其呈现的松散无序结构，也暗示其功能的复杂性。

Inositol 1,4,5-triphosphate (IP₃) is a very important second messenger in animals and plants, produced by the hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2). Extensive studies have shown that there exists an IP₃-Ca²⁺signalling pathway in animals and plants, in which IP₃ induces an increase of intracellular [Ca²⁺] ([Ca²⁺]_int). The mechanism of IP₃ receptor (IP₃R)-mediated Ca²⁺signalling in animal cells is relatively clear, but no IP₃-binding proteins have been found in plants so far. Our previous study showed that a cDNA fragment was isolated from the Arabidopsis cDNA library by functional screening in GSK cells (human embryonic kidney cells that knock out STIM1/2 and stably turn the GCaMP6 calcium ion indicator), which encodes a protein mediating extracellular [Ca²⁺] ([Ca²⁺]_ext)-triggered [Ca²⁺]_int increases in GSK cells after emptied the endoplasmic reticulum calcium with thapsigargin (TG). Sequence analysis showed that this cDNA fragment encodes the C-terminal of Phospholipase D regulated protein (PLDrp1), which is showed to bind phosphatidic acid (PA). Further research showed that PLDrp1 binds IP₃ within the physiological concentration range, and interacts with Annexin 4 (ANN4) to mediate flg22-induced [Ca²⁺]_int increases, which is vital for disease resistance response in Arabidopsis. Therefore, it is renamed as the IP₃ sensor (IP₃S).

This research mainly focused on the structure and functional relationship of IP₃S. Firstly, based on the primary sequence and predicted three-dimensional structure of IP₃S, it was found that the protein is an intrinsically disordered protein without a fixed domain or three-dimensional structure. We further found 14 conserved serial repeat sequences consisting of 12-15 amino acids from 110 amino acid (aa) to its C-end (381 aa), and the predicted short a-helix localized in the N-end (1-90 aa) of IP₃S. Therefore, we constructed two truncated proteins: IP₃S-Nt (1-90 aa) and IP₃S-Ct (91-381 aa), expressed and purified them using Escherichia coli strain Transeeta, respectively. By performing MicroScale Thermophoresis (MST) assay, we found that IP₃S-Ct binds IP₃, but IP₃S-Nt not. In addition, the binding Kd value of IP₃S-Ct to IP₃ is similar to the full-length IP₃S. By repeating the same strategy of vector construction, prokaryotic expression and purification, and MST assay, we found IBD-1 (90-143 aa) does not bind to IP3, but IBD-2 (144-181 aa) binds to IP₃ with the greater Kd value than that of full-length IP₃S.

Furthermore, the interaction of IP₃S and ANN4 was verified by IP₃S-Nt and IP₃S-Ct with ANN4 by using the Luciferase Complementation Imaging (LCI) Assay. Firstly, We constructed IP₃S-T1-180-cLUC and IP₃S-Ct-cLUC vectors, transiently transformed with ANN4-nLUC into tobacco leaves, respectively, and observed the fluorescence intensity of Luciferase. Then, we found the strong fluorescence in both combination of IP₃S-Nt-cLUC with ANN4-nLUC, and IP₃S-Ct-cLUC with ANN4-nLUC, and the fluorescence intensity was not significantly different. Secondly, the same strategy was used to verify the interaction of IP₃S fragments T1-180 (1-180 aa) and T181-381(181-381 aa) with ANN4, and there was no significant difference in the intensity of the interaction. Such results imply that there does not seem to be a specific region where IP3S interact with ANN4. In the future, we will detect the interaction of IP₃S with the different fragment of ANN4 by using the same method.

Finally, according to the characteristics of IP₃S binding to IP₃, we constructed IP₃ indicator by ligating the donor protein mTurquoise2 (the optimized mutant of blue fluorescent protein CFP) and the receptor protein mNeonGreen (the mutant of green fluorescent protein GFP) at the N- and C-terminals of IP₃S, respectively. Then, by performing the fluorescence resonance energy transfer (FRET) assay, we found no significant FRET change after IP₃S binding to IP₃, indicating that the conformation of IP₃S has no obvious change after binding to IP₃ because of its loose disordered structure.