【摘要】：向日葵列當(dāng)(Orobanche cumana Wallr.)為全寄生草本植物,可寄生在向日葵(Helianthus annuus L.)的根部,獲取水分和養(yǎng)分,從而對寄主向日葵產(chǎn)生不利影響,包括生長遲緩、產(chǎn)量與品質(zhì)下降,嚴(yán)重時還會引起植株死亡。同時,土壤中巨大的列當(dāng)種子庫、防治時間的難以準(zhǔn)確預(yù)測以及新的向日葵列當(dāng)生理小種的迅速出現(xiàn),使得目前對向日葵列當(dāng)?shù)姆乐畏椒@得不盡如人意。本實(shí)驗(yàn)以前期鑒定的向日葵抗性品種JY207和感性品種TK0407作為實(shí)驗(yàn)材料,在向日葵列當(dāng)侵染條件下,通過比對不同抗性向日葵品種之間植株生長、生理生化以及蛋白表達(dá)水平的差異,來探討向日葵列當(dāng)對向日葵的作用特性以及向日葵的抗性機(jī)制,為向日葵抗性品種的選育提供理論指導(dǎo)。同時,將感性品種向日葵種子進(jìn)行外源水楊酸的預(yù)處理,誘導(dǎo)向日葵產(chǎn)生防御反應(yīng),提高對向日葵列當(dāng)?shù)牡挚沽?為防治向日葵列當(dāng)提供了新的思路。主要研究結(jié)果如下:(1)向日葵列當(dāng)侵染以后,向日葵感性品種TK0409中活性氧自由基(ROS)的水平顯著提高,而在抗性品種JY207中,只有H2O2的含量略有上升而OH-和O2-的含量無明顯變化,同時,兩個品種中抗氧化物酶的活性均顯著提高,表明向日葵列當(dāng)?shù)那秩驹谙蛉湛袝餜OS的積累。然而,抗氧化物酶的活性增強(qiáng)程度在兩個品種中卻并不相同,POD和APX在兩個品種中活性提高的程度無差異,SOD的活性在TK0409中更高,而CAT和GR的活性在JY207中更高,這些結(jié)果表明對ROS的清除機(jī)制的激活程度,可能決定著向日葵的抗性能力。此外,受到向日葵列當(dāng)侵染以后,JY207中木質(zhì)素含量要顯著高于TK0409,表明木質(zhì)素的積累可能也有利于向日葵產(chǎn)生對列當(dāng)?shù)目剐浴６蛉湛捣置谖镏写龠M(jìn)列當(dāng)種子萌發(fā)的刺激物,其種類和含量的差異可能也與向日葵的抗性相關(guān)。(2)利用iTRAQ技術(shù),鑒定了兩個品種向日葵JY207和TK0409受到列當(dāng)侵染前后差異表達(dá)的蛋白。兩個品種均鑒定出超過3500個蛋白,其中抗性品種JY207包含706個差異表達(dá)的蛋白而感性品種TK0409中總共有866個差異表達(dá)的蛋白。這些蛋白的主要生化過程包括小分子代謝、生物合成、細(xì)胞內(nèi)氮復(fù)合代謝、運(yùn)輸過程、碳水化合物代謝過程和分解代謝過程。將差異表達(dá)蛋白比對到KEGG數(shù)據(jù)庫中,發(fā)現(xiàn)其主要代謝途徑包括碳代謝作用、氧化磷酸化、氨基酸合成、核糖體、RNA運(yùn)輸?shù)�。利用SRING數(shù)據(jù)庫來預(yù)測差異蛋白質(zhì)之間的相互作用,JY207中上調(diào)蛋白之間的互作主要涉及到核糖體、氧化磷酸化途徑以及蛋白酶相關(guān)蛋白,而下調(diào)蛋白之間的互作主要涉及到次生代謝途徑。TK0409中上調(diào)蛋白的互作主要涉及核糖體蛋白而下調(diào)蛋白主要涉及次生代謝途徑和碳水化合物代謝。對抗性相關(guān)的蛋白進(jìn)行鑒定,發(fā)現(xiàn)向日葵對向日葵列當(dāng)?shù)目剐钥赡苌婕暗蕉喾N信號途徑,是多種機(jī)制的綜合表現(xiàn),包括對列當(dāng)萌發(fā)刺激物的調(diào)節(jié)機(jī)制、對入侵寄生物的識別機(jī)制、防御相關(guān)蛋白的積累、木質(zhì)素的合成、有毒代謝產(chǎn)物的解毒機(jī)制以及對植物生長調(diào)節(jié)劑的調(diào)節(jié)作用。此外,決定品種為感性還是抗性,可能與植株受到侵染后抗性機(jī)制被激活的速度、程度以及效率有關(guān)。本研究中鑒定出來的蛋白質(zhì),為反向基因研究以及進(jìn)一步研究向日葵應(yīng)對列當(dāng)侵染的抗性機(jī)制提供理論參考,同時可用于輔助向日葵的抗性育種。(3)在列當(dāng)寄生的條件下,水楊酸在感性品種向日葵形成自身防御反應(yīng)的過程中發(fā)揮著重要作用。向日葵被列當(dāng)寄生后,其生物量急劇下降,而外源水楊酸對向日葵種子預(yù)處理后,這種下降趨勢有一定程度的減緩。在被向日葵列當(dāng)寄生的條件下,隨著外源水楊酸含量逐漸升高(從0.5到1 mM),與陽性對照組(未施水楊酸)相比,向日葵中H2O2和-OH的含量分別從7%和42%增加到14%和114%,而O2-的含量呈略微下降的趨勢,APX和CAT的活性在不同濃度(0.5和1 mM)的水楊酸作用下分別下降了 38%和18%、49%和42%。對于SOD活性,外源水楊酸并未對被寄生的向日葵根部產(chǎn)生影響。被向日葵列當(dāng)寄生后,大部分基因包括PR基因,一些抗氧化物酶相關(guān)基因以及植物激素相關(guān)基因都呈上調(diào)趨勢,而與此不同的是,pal、cHs和nPr1的相對表達(dá)量分別下降了 55%、56%和34%,以及Cu/Zn-SOD、GST和hsr的相對表達(dá)量并無顯著變化。在被向日葵列當(dāng)寄生的條件下,外源水楊酸的使用提高了大部分基因的相對表達(dá)量,而C4T、lox、PR5-1、HACS.1和chs等基因則無明顯變化。這些研究結(jié)果表明外源水楊酸通過調(diào)節(jié)ROS水平、抗氧化酶系統(tǒng),誘導(dǎo)NPR1、病程相關(guān)基因以及其他防御相關(guān)基因的表達(dá),使感性品種向日葵形成系統(tǒng)獲得性抗性和超敏反應(yīng)來緩解向日葵列當(dāng)?shù)那秩尽?br/>[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S453

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