P450酶功能化光纤微电极的开发及植物活体一氧化氮和脱落酸的光电检测应用研究

Recently, how to detect signaling molecules in cells in situ is the hot topic in life sciences. Nitric oxide and abscisic acid are both extremely important signaling molecules involved in many physiological processes in plants. The accurately determination and spatial patterns are the fundmentals to study the mechanism of signal transduction and interaction. It has been reported that nitric oxide and abscisic acid are separately catalyzed by cytochrome P450 55B1 and 707A3. This project is to investigate the combination of the recognition and catalytic ability of cytochrome P450s, enrichment capacity of nanoparticles and photoelectric property and in vivo detection ability of optical fiber microelectrodes with an aim to develop novel optical/electrochemical methods to detect nitric oxide and abscisic acid in plants in vivo with high selectivity and high sensitivity. Firstly, cytochrome P450s with high activity are to be created and isolated. Then the development of cytochrome P450s and nanoparticles functionalized optical fiber microelectrodes is to be performed. Finally, the characteristic of molecular recognition and the mechanism of optical/electrochemical response of functionalized optical fiber microelectrodes are to be studied to attain new optical/electrochemical theories for biosensing nitric oxide and abscisic acid. New methods are to be achieved to detect nitric oxide and abscisic acid in-situ and real time ultimately and these methods are to be applied to detect nitric oxide and abscisic acid in plants in vivo simulanteously. The implementation of this project not only can develop a new method to detect nitric oxide and abscisic acid in situ for the study on the mechanism of signal transduction and interaction, but also can further extend the development of new systems to detect signaling molecules in plants in vivo.

细胞内信号分子的原位监测是当前生命科学研究热点。一氧化氮和脱落酸是两种重要的植物信号分子，其含量测定及时空分布是研究其信号传导及互作机制的基础。本项目旨在将细胞色素P450酶55B1和707A3对一氧化氮和脱落酸的专一识别催化能力、纳米粒子的富集能力和光纤微电极的活体光电分析功能相结合，创建高灵敏度和高选择性的一氧化氮和脱落酸光电生物传感新方法。首先创制高活性的P450酶，进而研究纳米粒子和P450酶功能化光纤微电极的制备，最后利用P450酶的生物催化特性研究P450酶功能化光纤微电极的分子识别特性和光电响应机制，获得一氧化氮和脱落酸光电生物传感新理论，建立一氧化氮和脱落酸的实时、原位、动态检测新方法，并应用于植物活体一氧化氮和脱落酸的同时光电检测。本项目的实施不仅能为一氧化氮和脱落酸的信号传导及互作机制研究提供一种新的原位检测方法，而且有利于构筑更加广泛的植物活体信号分子检测新体系。

基于细胞色素P450酶55B1对一氧化氮专一识别和催化能力制备了高灵敏度和高选择性的一氧化氮光电生物传感器。首先在大肠杆菌中成功表达了P450 55B1，纯化后的蛋白样CYP55B1含量为133.85 nM/L。NO的加入引起P450 55B1的卟啉铁的荧光强度逐渐增加。 当NO浓度低于22.5 μM时，荧光强度的变化与NO浓度呈线性关系：ΔF = 0.0675 + 0.15 CNO（μM），该荧光传感器抗干扰能力强。采用戊二醛和牛血清白蛋白交联法将细胞色素P450 55B1 固定在热解石墨电极表面，构建了一氧化氮( NO)电化学生物传感器，细胞色素P450 55B1实现了直接电子传递，氧化还原峰电位位于－0．355 V 和－0．385 V，细胞色素P450 55B1 对NO具有电催化活性，其催化还原峰位于－0．85 V， NO的检测线性范围为14．4～108 μM，检测限为10.47 μM，该生物传感器具有良好的稳定性和选择性，并成功应用于拟南芥原生质体的NO的检测。.基于细胞色素P450 707A3酶对脱落酸专一识别催化能力开发了高灵敏度和高选择性的脱落酸电化学生物传感器。首先在大肠杆菌中成功表达了P450 707A3，纯化后的蛋白样CYP707A3含量为1.63 nM/L。利用双十六烷基磷酸将细胞色素P450 707A3固定在热解石墨电极表面， 细胞色素P450 707A3实现了直接电子传递，氧化还原峰电位位于－0.50 V 和－0．58 V，细胞色素P450 707A3对脱落酸电催化氧化，氧化电流与脱落酸浓度线性范围为5-30 nM，检测限为4.85 nM，该生物传感器应用于拟南芥原生质体脱落酸的检测。.基于水稻脱落酸受体PYL2与蛋白磷酸酶PP2C相互作用，建立脱落酸BRET检测新方法。构建了八个BRET信号模块，以大肠杆菌BL21为宿主菌，表达了八个融合蛋白，通过BRET信号模块优化，得到生物发光效率最高的BRET信号模块组合。利用最优的BRET体系检测脱落酸，检测的线性范围为0.005～0.1 µM，线性回归方程y = 0.53344x + 0.06597（R² = 0.88632）,检测限（LOD）3.8 nM，并考察了BRET体系对脱落酸的专一性。最后将该体系应用于水稻叶片脱落酸含量检测。