基于脂肪酸代谢关键酶控制柑桔黄龙病的分子机制的研究

Citrus Huanglongbing (HLB) is one of the most devastating diseases that have menaced citrus production at home and abroad, the key scientific problems to control the pathogen need to be resolved urgently. This project will integrate Structural Biology and the research of resistance disease in plant, the key enzymes of the fatty acid metabolism would be used as the point of penetration in Candidatus Liberibacter asiaticus. By cloning the genes of coding the enoyl-acyl carrier protein (ACP) reductase I (FabI), β-hydroxyacyl (ACP) dehydrase (FabZ), 3-oxoacyl (ACP) synthesis (FabH) and 3-oxoacyl (ACP) reductase (FabG), and constructing the protokaryon expression vectors, the proteins would be expressed, isolated and purified, crystals were prepared and analysesed, and sited directly mutation would be performed, the combination way and active sites among the protein, prosthetic group and inhibitor would be elucidated. The inhibitors would be selected guiding by structure information, the spectrograph, surface plasmon resonance systems(SPR) and isothermal titration calorimetry (ITC) will be used for evaluating the characteristics of competitive and noncompetitive, the speed of connection and dissociation, the specificity and selectivity of the combination between the enzyme and inhibitor, solubility and membrane permeability of inhibitor. To find the optimal inhibitors which can blocking-up fatty acid metabolism by the test in the indicative plant of HLB, To elucidate accordingly the molecular mechanism of control the Candidatus Liberibacter asiaticus and proved the thinking and theory foundation for the control of the unable to be cultured bacteria.

柑桔黄龙病对国内外柑桔产业造成极大的威胁，解决控制病原关键的科学问题十分迫切。本课题将结构生物学与植物抗病研究有机结合，以韧皮部杆菌脂肪酸代谢关键酶为切入点，通过克隆编码脂肪酸代谢的关键酶：烯脂酰ACP还原酶(FabI)及β-酮脂酰ACP合成酶（FabH）、羟脂酰ACP脱水酶(FabZ)和β-酮脂酰基ACP还原酶(FabG)的基因，构建表达载体，在E.coli中进行原核表达，通过蛋白质分离、纯化、晶体制备和解析及位置直接突变，阐明蛋白质、辅基及抑制剂的结合方式和活性位点；通过结构信息指导抑制剂筛选，利用光谱仪、表面等离子体共振、等温滴定量热仪综合评价酶与抑制剂的竞争与非竞争性，结合和解离速度，结合的特异性、选择性、抑制剂的溶解性和膜透性，并对黄龙病敏感的指示植物进行试验，为发现阻断脂肪酸代谢最佳的抑制剂提供线索。从而阐明控制韧皮部杆菌的分子机制，为难培养细菌的控制提供新思路和理论依据。

柑桔黄龙病对国内外柑桔产业造成极大的威胁，解决控制病原关键的科学问题十分迫切。本课题将结构生物学与植物抗病研究有机结合，以韧皮部杆菌脂肪酸代谢关键酶为切入点，开展了烯酰-ACP还原酶（FaI)和羟脂酰ACP脱水酶（FabZ)蛋白表达、纯化；解析了FabI，FabI-NAD和FabZ三种蛋白质的晶体，并在 PDB上公布了晶体学数据;对FabZ蛋白溶液的聚集状态进行了小角分析，确定了FabZ蛋白为六聚体；验证了柑桔黄龙病FabZ蛋白与ACP互作关系;开展了柑桔黄龙病FabH和FabG蛋白表达、纯化研究；利用表面等离子体共振技术（SPR）、微尺度热泳（MST）和生物膜层表面干涉技术(FLI)检测了蛋白质FabI与多种小分子的互作关系,筛选出5种小分子；开展了小分子抑制剂在柑桔接穗和苗木上控制柑桔黄龙病的试验,其中，3种小分子对接穗的黄龙病病原脱除有效，2种小分子对苗木黄龙病控制有效;研究了抑制剂控制柑桔黄龙病机制，并进行了抑制剂处理柑桔寄主后的转录组测序分析,明确了起关键作用的调控基因。