向日葵应对列当侵染的作用机理研究及外源水杨酸的缓解效应

发布时间：2018-09-12 10:52

【摘要】：向日葵列当(Orobanche cumana Wallr.)为全寄生草本植物,可寄生在向日葵(Helianthus annuus L.)的根部,获取水分和养分,从而对寄主向日葵产生不利影响,包括生长迟缓、产量与品质下降,严重时还会引起植株死亡。同时,土壤中巨大的列当种子库、防治时间的难以准确预测以及新的向日葵列当生理小种的迅速出现,使得目前对向日葵列当的防治方法显得不尽如人意。本实验以前期鉴定的向日葵抗性品种JY207和感性品种TK0407作为实验材料,在向日葵列当侵染条件下,通过比对不同抗性向日葵品种之间植株生长、生理生化以及蛋白表达水平的差异,来探讨向日葵列当对向日葵的作用特性以及向日葵的抗性机制,为向日葵抗性品种的选育提供理论指导。同时,将感性品种向日葵种子进行外源水杨酸的预处理,诱导向日葵产生防御反应,提高对向日葵列当的抵抗力,为防治向日葵列当提供了新的思路。主要研究结果如下:(1)向日葵列当侵染以后,向日葵感性品种TK0409中活性氧自由基(ROS)的水平显著提高,而在抗性品种JY207中,只有H2O2的含量略有上升而OH-和O2-的含量无明显变化,同时,两个品种中抗氧化物酶的活性均显著提高,表明向日葵列当的侵染在向日葵中会引起ROS的积累。然而,抗氧化物酶的活性增强程度在两个品种中却并不相同,POD和APX在两个品种中活性提高的程度无差异,SOD的活性在TK0409中更高,而CAT和GR的活性在JY207中更高,这些结果表明对ROS的清除机制的激活程度,可能决定着向日葵的抗性能力。此外,受到向日葵列当侵染以后,JY207中木质素含量要显著高于TK0409,表明木质素的积累可能也有利于向日葵产生对列当的抗性。而向日葵根系分泌物中促进列当种子萌发的刺激物,其种类和含量的差异可能也与向日葵的抗性相关。(2)利用iTRAQ技术,鉴定了两个品种向日葵JY207和TK0409受到列当侵染前后差异表达的蛋白。两个品种均鉴定出超过3500个蛋白,其中抗性品种JY207包含706个差异表达的蛋白而感性品种TK0409中总共有866个差异表达的蛋白。这些蛋白的主要生化过程包括小分子代谢、生物合成、细胞内氮复合代谢、运输过程、碳水化合物代谢过程和分解代谢过程。将差异表达蛋白比对到KEGG数据库中,发现其主要代谢途径包括碳代谢作用、氧化磷酸化、氨基酸合成、核糖体、RNA运输等。利用SRING数据库来预测差异蛋白质之间的相互作用,JY207中上调蛋白之间的互作主要涉及到核糖体、氧化磷酸化途径以及蛋白酶相关蛋白,而下调蛋白之间的互作主要涉及到次生代谢途径。TK0409中上调蛋白的互作主要涉及核糖体蛋白而下调蛋白主要涉及次生代谢途径和碳水化合物代谢。对抗性相关的蛋白进行鉴定,发现向日葵对向日葵列当的抗性可能涉及到多种信号途径,是多种机制的综合表现,包括对列当萌发刺激物的调节机制、对入侵寄生物的识别机制、防御相关蛋白的积累、木质素的合成、有毒代谢产物的解毒机制以及对植物生长调节剂的调节作用。此外,决定品种为感性还是抗性,可能与植株受到侵染后抗性机制被激活的速度、程度以及效率有关。本研究中鉴定出来的蛋白质,为反向基因研究以及进一步研究向日葵应对列当侵染的抗性机制提供理论参考,同时可用于辅助向日葵的抗性育种。(3)在列当寄生的条件下,水杨酸在感性品种向日葵形成自身防御反应的过程中发挥着重要作用。向日葵被列当寄生后,其生物量急剧下降,而外源水杨酸对向日葵种子预处理后,这种下降趋势有一定程度的减缓。在被向日葵列当寄生的条件下,随着外源水杨酸含量逐渐升高(从0.5到1 mM),与阳性对照组(未施水杨酸)相比,向日葵中H2O2和-OH的含量分别从7%和42%增加到14%和114%,而O2-的含量呈略微下降的趋势,APX和CAT的活性在不同浓度(0.5和1 mM)的水杨酸作用下分别下降了 38%和18%、49%和42%。对于SOD活性,外源水杨酸并未对被寄生的向日葵根部产生影响。被向日葵列当寄生后,大部分基因包括PR基因,一些抗氧化物酶相关基因以及植物激素相关基因都呈上调趋势,而与此不同的是,pal、cHs和nPr1的相对表达量分别下降了 55%、56%和34%,以及Cu/Zn-SOD、GST和hsr的相对表达量并无显著变化。在被向日葵列当寄生的条件下,外源水杨酸的使用提高了大部分基因的相对表达量,而C4T、lox、PR5-1、HACS.1和chs等基因则无明显变化。这些研究结果表明外源水杨酸通过调节ROS水平、抗氧化酶系统,诱导NPR1、病程相关基因以及其他防御相关基因的表达,使感性品种向日葵形成系统获得性抗性和超敏反应来缓解向日葵列当的侵染。
[Abstract]:Orobanche Cumana Wallr. is a fully parasitic herb. It can parasitize on the roots of Helianthus annuus L. to obtain water and nutrients. It has adverse effects on the host sunflower, including slow growth, decreased yield and quality, and even causes plant death in serious cases. At the same time, the huge soil column when the seed bank, It is difficult to accurately predict the control time and the rapid emergence of new physiological races in sunflower, which makes the current control methods of sunflower ledang unsatisfactory. The resistance varieties JY207 and susceptible varieties TK0407 identified earlier in this experiment were used as experimental materials and compared with each other under the condition of sunflower ledang infection. The differences of plant growth, physiology, biochemistry and protein expression among the same resistant sunflower cultivars were studied to explore the action characteristics and resistance mechanism of Lidang in order to provide theoretical guidance for the breeding of resistant sunflower cultivars. The main results were as follows: (1) The level of reactive oxygen species (ROS) in the susceptible Sunflower Variety TK0409 was significantly increased after the infection, while the content of H2O2 in the resistant variety JY207 was only slightly increased. The activity of antioxidant enzymes in both cultivars was significantly increased, suggesting that ROS accumulation was caused by the infection of sunflower ledang. However, the activity of antioxidant enzymes was not the same in the two cultivars, and the activity of POD and APX increased in the two cultivars. There was no difference in SOD activity between JY207 and TK0409, whereas CAT and GR activity were higher in JY207. These results indicated that the activation of ROS scavenging mechanism might determine the resistance of sunflower. In addition, the lignin content of JY207 was significantly higher than that of TK0409 after infection by sunflower row, indicating that lignin accumulation might also be beneficial. (2) The differentially expressed proteins of two sunflower varieties JY207 and TK0409 were identified by iTRAQ technique. More than 3 500 proteins were identified, including 706 differentially expressed proteins in resistant variety JY207 and 866 differentially expressed proteins in susceptible variety TK0409. The main biochemical processes of these proteins include small molecule metabolism, biosynthesis, intracellular nitrogen complex metabolism, transport, carbohydrate metabolism and catabolism. By comparing the differentially expressed proteins with KEGG databases, we found that their main metabolic pathways include carbon metabolism, oxidative phosphorylation, amino acid synthesis, ribosome, RNA transport, etc. The interaction between the up-regulated proteins and the down-regulated proteins is mainly related to the secondary metabolic pathway. The up-regulated proteins in TK0409 are mainly related to ribosomal proteins, while the down-regulated proteins are mainly related to the secondary metabolic pathway and carbohydrate metabolism. It may involve a variety of signaling pathways and is a comprehensive manifestation of a variety of mechanisms, including the regulation of germination stimuli, the recognition of invasive parasites, the accumulation of defense-related proteins, the synthesis of lignin, the detoxification of toxic metabolites, and the regulation of plant growth regulators. The proteins identified in this study can provide theoretical reference for reverse gene research and further study on the resistance mechanism of sunflower to Lidang infection, and can also be used to assist the resistance breeding of sunflower. Under parasitic conditions, salicylic acid plays an important role in the formation of self-defense responses in susceptible sunflower cultivars. When sunflower is listed as parasitic, its biomass decreases sharply, while the decline trend is slowed down to a certain extent after seed pretreatment with exogenous salicylic acid. Compared with the positive control group (without salicylic acid), the content of H2O 2 and - OH in sunflower increased from 7% and 42% to 14% and 114% respectively, while the content of O2 - decreased slightly. The activities of APX and CAT decreased by 38% and 18%, 49% respectively under salicylic acid of different concentrations (0.5 and 1 mM). And 42%. For SOD activity, exogenous salicylic acid had no effect on the root of the parasitized sunflower. After being parasitized by sunflower, most genes including PR gene, some antioxidant enzyme-related genes and plant hormone-related genes were up-regulated, but the relative expression of pal, cHs and nPr1 decreased by 55% respectively. The relative expression levels of Cu/Zn-SOD, GST and HSR did not change significantly. The use of exogenous salicylic acid increased the relative expression levels of most genes while C4T, lox, PR5-1, HACS.1 and CHS did not. Antioxidant enzymes induce the expression of NPR1, pathogenesis-related genes and other defense-related genes, and form systemic acquired resistance and hypersensitivity in susceptible sunflower varieties to alleviate the infection of Lidang.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S453

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