【作者】 李杰；

【导师】 刘树生； 王晓伟；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2015， 博士

【摘要】 烟粉虱(Bemesia tabaci)是由至少36个隐种组成的隐种复合体,其中的一些隐种如MEAM1和MED由于其繁殖能力强、寄主适应范围广、抗药性强等特点近年来随着贸易活动的往来快速地入侵到世界各地。它们以持久性可循环的方式传播双生病毒,并与其进化出独特的关系。烟粉虱的暴发通常伴随着双生病毒病的流行。双生病毒对寄主植物的基因表达和生理功能的调控可能影响烟粉虱的种群动态,进而又影响病毒本身的传播。茉莉酸信号通路(JA signaling)及其调节的次生代谢物质(如芥子油苷和萜类化合物等)在植物应对病原物及昆虫取食过程中起着重要作用。本文先从烟粉虱的角度出发探讨了植物JA防御途径对烟粉虱适合度的影响及其基因表达的调控；然后探讨了重要农业经济作物番茄在应对烟粉虱取食、病毒感染以及两者共同侵染时的基因表达变化；最后以模式植物拟南芥为材料,探讨烟粉虱及病毒致病因子pC1在拟南芥芥子油苷生成中的影响以及这种影响对烟粉虱适合度的作用。研究结果如下：1) JA防御途径对烟粉虱基因表达及适合度的影响通过第二代测序技术,从转录水平上揭示了MEMA1烟粉虱应对植物JA防御反应的分子机制。烟粉虱在JA激活的烟草上取食3天后,共有242条基因上调,450条基因下调。对这些基因的分析结果表明,烟粉虱的初级代谢过程如糖代谢、氨基酸代谢、脂代谢等等都被显著地下调；与能量代谢有关的基因都被显著下调,说明能量代谢很可能受到抑制。然而与烟粉虱解毒相关的基因都显著地上调,说明烟粉虱解毒活动增强。另外,结果显示,与烟粉虱细胞周期、神经性疾病等有关的基因都受到显著地调节。这些结果表明烟粉虱主动调节其基因表达来降低植物防御对其的伤害,却也可能仍然受到植物抗性物质的毒害作用,导致烟粉虱在JA途径激活的烟草上死亡率升高。2)番茄应对烟粉虱取食、病毒感染以及烟粉虱-病毒共同侵染的分子机制通过第二代测序技术,分析了番茄在受到烟粉虱取食、病毒感染以及烟粉虱-病毒共同侵染条件下的基因表达。三种处理得到的差异表达基因分别为324条、686条和996条,共同感染对番茄基因表达的影响最为明显,不同处理间调节的基因有部分重叠也有各处理特异差异表达的基因,并且重叠的基因大多都有着相同的调节模式。病毒感染及共同感染显著地抑制了番茄的光合作用及苯丙素和类黄酮代谢过程,可能与病毒感染引起番茄植物黄化有关。同时,许多与细胞壁及碳代谢有关的基因都受到显著地调节。番茄免疫反应相关的许多抗性基因以及病程相关基因被显著上调,说明番茄可能通过基础免疫以及基因对基因抗性来识别和抵御烟粉虱取食和病毒感染。在对植物激素的分析中,发现JA途径相关基因受到病毒感染和共同侵染时的抑制,而与生长有关的激素如生长素、细胞分裂素、脱落酸、赤霉素等受到更为显著地调节,这些可能与病毒感染及昆虫取食引起植物生理异常如矮化、叶片卷曲等有关。另外有许多差异表达基因没有功能注释或者其在植物-昆虫-病毒互作中的研究很少,如组蛋白相关基因。3)次生代谢物芥子油苷在烟粉虱-植物-病毒三者互作中的作用通过对烟粉虱处理以及转pC1基因植株芥子油苷含量的测定,发现烟粉虱的取食能显著诱导拟南芥芥子油苷的生成,而pC1基因能显著地抑制拟南芥芥子油苷的生成。莲座叶中脂肪族芥子油苷含量在生长初期占主要部分,随着拟南芥的生长而逐渐降低,而吲哚族芥子油苷含量保持稳定并在后期成为叶片中主要的芥子油苷。生物测定结果显示,芥子油甘含量降低没有显著提高烟粉虱产卵量,但吲哚族芥子油苷的过量表达能显著影响烟粉虱的产卵量及发育速率；βC1基因通过影响JA途径而提高烟粉虱的适合度,次生代谢物芥子油苷及萜类等物质都可能参与到该过程。综上所述,本研究分别探索了番茄应对烟粉虱取食、病毒感染以及烟粉虱-病毒共同侵染时的分子机制以及植物JA防御途径对烟粉虱基因表达的影响;并且探讨了次生代谢物芥子油苷在烟粉虱-植物-病毒三者互作中的作用。这些研究结果有助于我们更好地理解这三者的关系,并为进一步研究抗性基因和研发防治二者共同为害的新方法提供线索。更多还原

【Abstract】 The whitefly (Bemisia tabaci) is a species complex comprised of at least 36 cryptic species, of which some species such as MEAM1 and MED have been spreading around the world in the past 30 years because of their strong reproductive ability, broad host range, and high resistance to insecticides. They have evolved a specific relationship with begomoviruses, which they transmit in a circulative, persistent manner. Outbreaks of whiteflies are often followed by pandemics of begomoviruses. The reprogramming of gene expression and the regulation of plant physiology may influence the population dynamics of whiteflies and in turn the spread of the virus itself. Jasmonate signaling pathway and the secondary metabolite production that it regulates, such as glucosinolates, plays a vital role in plant’s defense against pathogen infection and insect herbivory.In the present study, we firstly examined the effect of plant JA defenses on the gene expression of whiteflies and the performance of whiteflies on JA-elicited tobacco plants; then we used RNA-seq to investigate the gene expression profile of tomato to whitefly infestation, virus infection and virus-whitefly co-infestation; and lastly we used the model plant Arabidopsis thaliana to investigate the influence of whitefly feeding and begomovirus pathogenicity factor βC1 gene on the production of glucosinolates and the role of glucosinolates on the performance of whiteflies in this context. The results are summarized as follows:1. The influence of JA defense pathway on the gene expression and fitness of whitefliesWe conducted a comprehensive investigation on the responses of the MEAM1 whitefly to plant JA-regulated defenses, using RNA-seq technology. After feeding for 3 days on JA-elicited tobacco, whiteflies have 242 genes up-regulated and 450 genes down-regulated. Further analysis of these differentially expressed genes (DEGs) revealed that the primary metabolism such as carbon hydrate metabolism, amino acid metabolism, lipid metabolism etc. were all significantly repressed, and genes associated with energy metabolism were all down-regulated. Meanwhile, nearly all the DEGs involved with detoxification were up-regulated, suggesting that the primary metabolism including energy production in whiteflies was repressed while it still managed to increase the detoxification activity. On the other hand, genes involved with cell cycle, and neural diseases were also significantly regulated. All these results indicate that while whiteflies actively detoxify the plant defenses to reduce the damage of plant defensive chemicals on them, plant defensive chemicals may still have some toxic effect on whiteflies leading to higher mortality of whiteflies on JA-elicited tobacco.2. Responses of tomato to whitefly feeding, virus infection and virus-whitefly co-infestationWe performed digital gene expression profile to examine the molecular responses of tomato plants to whitefly feeding, virus infection and virus-whitefly co-infestation. The three treatments resulted in 324,686 and 996 DEGs, respectively. Among the three treatments, co-infestation has the most pronounced effect on tomato. There were overlapping of DEGs between treatments as well as treatment-specific DEGs, and a large proportion of the overlapping genes have the same transcript pattern. Virus infection and co-infestation significantly suppressed photosynthesis rates and Phenylpropanoid/ Flavonoid biosynthesis, which might be a direct sign of perturbation of pigment biosynthesis leading to chlorosis of leaves. And also many genes involved in cell wall regulation and carbon fixation, both of which might be involved in plant pathogen resistance, were significantly influenced. Resistance genes and pathogenesis-related genes were up-regulated in these treatments, indicating that tomato might use basal resistance as well as gene-to-gene resistance to recognize and defend against virus and whiteflies. In the analysis of genes involved with plant hormones, we found that genes in JA pathway were repressed in plants infected by the virus and plants co-infested by the virus and whitefly. Surprisingly, hormones involved in plant growth, such as indole-3-acetic acid, abscisic acid, cytokinin, gibberellin and brassinosteroid, were more profoundly influenced in these three treatments. Effects on these plant growth hormones might be involved in the stunting, reduction of leaf size, and upward cupping/curling phenotype in begomovirus infected tomato. Besides, there are a whole set of genes that were significantly regulated but have yet no functional annotation or clear role in virus-plant-insect interactions, such as the histone related genes.3. The role of glucosinolates in whitefly-hostplant-virus interactionWe tested the content of glucosinolates in Arabidopsis plants in response to whitefly feeding and glucosinolates content in 35S:HA-βC1/Co1-0 transgenic plants. We found that whitefly feeding induced the production of glucosinolates while βC1 gene repressed the production of glucosinolates. In Arabidopsis vegetative growth, the aliphatic glucosinolates in rosette leaves were the majority at the beginning and started to decline as the plants grew, while the content of indole glucosinolates remained little changed or even increased as the plants grew. Whitefly performance bioassay showed that lower level of glucosinolates did not improve the fecundity significantly. However, in an indole glucosinolate over-production mutant, whiteflies have significantly reduced fecundity and growth rate. The βC1 gene improved whitefly performance, probably via the repression of JA pathway and secondary metabolites such as glucosinolates and terpenes.更多还原