基于生物素蛋白连接酶特异性控制柑桔黄龙病病原的机制的研究

黄龙病是柑桔上难以控制的毁灭性病害，解决控制黄龙病病原关键的科学问题极为迫切。在前期研究的基础上，本课题组提出以韧皮部杆菌代谢中的关键酶——生物素蛋白连接酶（BPL）为切入点来控制黄龙病的新途径。首先克隆BPL基因，经过表达、纯化、结晶和结构解析，揭示BPL和生物素羧基载体蛋白亚基(BCCP)的晶体以及生物素反应中间体与BPL的复合晶体的结构特点；阐明由生物素或类似物结合诱导BPL产生有序化构象变化的规律，获得生物素反应中间体结合到BPL高度保守loop环上氨基酸活性位点的重要信息；采用表面等离子体共振等方法，分析BPL与抑制剂发生酶促反应的酶动力学规律，从分子和原子水平阐明BPL控制韧皮部杆菌的机制；再从多种特定结构生物素中间体小分子中，筛选对HLB有效的特异性抑制剂，并用敏感指示植物长春花验证其效果，为防治黄龙病开辟有效新方法奠定基础，也为所有其它难以培养细菌的防控研究提供理论依据。

Huanglongbing is a destructive disease，which is hard to control in citrus, the key scientific problems to control the HLB pathogen need to be resolved urgently. Basing on the previous research, a new pathway of controlling HLB is proposed with regulation the key enzyme -- biotin protein ligase (BPL) as an entry point during the bacterium metabolic pathway of Candidatus L. asiaticum. At first, adopting gene cloning, protein expression, purification, and crystal preparation and 3D structure analysis, the crystallography of biotin protein ligase, biotin carboxyl carrier protein, and complex crystal of biotinyl-5′-AMP-BPL will be characterized; The ordered binding mechanism by biotin or biotin analogs binding and induction the conformational change of biotin protein ligase (BPL) will be clarified, the amine acid active sites in conservative loop of BPL, which binding to biotinyl-5’-AMP will be found; and then, the enzyme kinetics between BPL and inhibitors by Surface Plasmon Resonance etc. will be analysesed. It will interpret that the molecular and atom mechanism to control Candidatus L. asiaticum basic on BPL; The sensitive indicating plant periwinkle test will carried out for verification the optimum inhibitors from the special structure biotinyl-5’-AMP analogs small molecular to control the Huanglongbing in citrus. Thereby, this project will provide the important theory base for opening up a new effective method of controlling HLB and antisepticise research against all the other diseases, which are caused by the uncultured bacterium.