槲皮素对Hsf的作用机理研究及具有病毒抑制活性的槲皮素类似物的筛选

A large number of experiments in vivo indicated quercetin had an antiviral activity against many plant viruses. The study of the action mechanism indicated quercetin can down-regulate the expression of Hsp, influence the formation of macromolecular protein complexes of plant virus, and then disturb disassembly, replication, movement, or assembly of viral particles. So, the study of quercetin has displayed a promising prospect for antiphytoviral agents. In this research, SMRT DNA sequencing was used to obtain Hsf gene of Nicotiana benthamiana under the stress conditions of high temperature or virus infection. The action mechanism of quercetin against Hsf was studied in the abundance of Hsf mRNA, phosphorylation of Hsf, the formation of Hsf polymer, binding between quercetin and trimerization domain of Hsf, binding of Hsf and Hsp promoter treated by quercetin. Meanwhile, in order to establish a reasonable bioactivity model, the effective relation was further concluded from treatment dosage, treatment time, viral kind, tissue or position, and the abundance of Hsp mRNA through the study object of quercetin and Nicotiana benthamiana. Hydroxy on C3 and C3’ of quercetin was substituted with aglycon, salicylic acid, and isonicotinic acid et al, and then evaluated about their anti-viral activity. Based on the structure-activity relationship, quercetin was further embellished and modified for enhancing the antiviral activity. Our studies would contribute to find the anti-viral compounds with higher activity based on the novel action mechansim.

大量活体试验表明槲皮素对多种植物病毒均有抑制活性。作用机理表明槲皮素可下调植物寄主Hsp表达，影响植物病毒MRC形成，干扰病毒脱衣壳、复制、运动或组装等过程。因此，槲皮素在植物病毒剂研究方面显示出潜在的应用前景。本项目拟采用三代全长转录组测序方法获得本氏烟在高温胁迫或病毒侵染条件下的Hsf基因。并从Hsf的mRNA丰度、磷酸化及多聚体形成，以及槲皮素与Hsf的Trimerization结合、槲皮素干扰Hsf与Hsp启动子的结合等环节研究其作用Hsf的机理。同时，为建立合理的活性筛选模型，槲皮素和本氏烟被作为研究对象，作用剂量、处理时间、病毒种类、组织部位、Hsp表达间的效应关系被进一步总结。试验拟针对槲皮素C3、C3’羟基进行各种基团的取代修饰，评价其抗病毒活性。基于构效关系的结果，槲皮素被进一步改造修饰，以提升抑制病毒的活性。本项目有望基于新颖作用机制发现新型高活性抗植物病毒化合物。

针对TMV等植物病毒和Fusarium oxysporum等真菌筛选有较高防治活性的槲皮素衍生物，发现7,4′-Di-O-甲基槲皮素等对TMV等病毒具有较强抑制活性，槲皮素-3-O-(2′-O-没食子酰基)-β-D-吡喃葡萄糖苷等对Fusarium oxysporum等真菌有较强的抑菌活性。采用转录组研究热激、槲皮素处理对本氏烟的DEmRNAs的影响，采用GO和KEGG进行DEmRNAs的注释，分析热激、TMV侵染及槲皮素对Hsp和Hsf基因表达的影响、对本氏烟Hsp和Hsf磷酸化的影响，发现槲皮素下调热热激诱导Hsp70 mRNA较为显著。发现42℃热激或TMV侵染，Hsf A和Hsf B表达上调，磷酸化Hsf B和Hsf C显著上调，采用槲皮素及热激，槲皮素可抑制Hsf磷酸化。热激和槲皮素处理对于MAPKKKe2等3个激酶基因表达没有显著影响，说明热激和槲皮素仅通过酶促活性调控Hsf磷酸化。热激或TMV侵染的Hsf A有明显聚合趋势，槲皮素对Hsf A聚合影响不明显。通过构建将DNA-binding模体、寡聚化结构域、核定位序列3个部分的本氏烟转基因突变体，研究发现热激能上调Hsf突变体，而槲皮素可下调热激对Hsf突变体的上调表达，说明槲皮素对Hsf的基因表达区上调表达没有明显影响。采用RNA-seq方法研究槲皮素及其衍生物对本氏烟Hsp70 mRNA的表达影响，发现7,4′-Di-O-甲基槲皮素对Hsp70 mRNA的调控最为明显。研究Hsf A2和Hsf B3与其他蛋白互作，发现与Hsf A2和Hsf B3互作蛋白不完全一致，其中MBF均与两个Hsf有互作；同时，Hsf A2与ROF、CRK有互作。YTHS验证表明，Hsf A2和MBF、Hsf B3与MBF存在互作。