【作者】 王军节；

【导师】 毕阳；

【作者基本信息】 甘肃农业大学 ， 作物保护， 2013， 博士

【摘要】 由真菌侵染引起的病害是导致新鲜果蔬采后烂损的重要原因。目前的控制手段主要是使用化学杀菌剂，但存在药物残留、环境污染以及诱导病原物产生抗药性等问题而逐渐受到限制。诱导抗病性是一种新型安全的采后病害控制措施，可取代或减少化学杀菌剂的使用，可溶性硅处理可通过诱导抗性来减轻多种果实采后病害的发生。本文以“早酥”梨、“富士”苹果和“玉金香”甜瓜为材料，采用采后病理学、生物化学和蛋白质组学等方法，研究了硅酸钠处理对三种果实采后主要病害的控制效果，探讨了处理对果实过氧化氢积累或代谢的影响，分析了处理对甜瓜蛋白质组学的影响。结果表明：1.采后100mmol/L硅酸钠处理能有效控制梨果实损伤接种Penicilliumexpansum的病斑扩展，处理提高了果实POD活性,抑制了APX和CAT活性。采用0.1mmol/L硅酸钠结合质膜过氧化氢阻断剂DPI接种法处理时发现，DPI处理减弱了硅酸钠对梨果实NOX活性和H2O2积累的诱导，从而降低了硅酸钠对果实青霉病的控制效果。2.200mmol/L硅酸钠处理可有效降低苹果损伤接种P. expansum的青霉病发病率和病斑面积。处理增加了果实SOD和NOX活性，降低了CAT和APX活性，导致了O2-和H2O2的积累。硅酸钠结合DPI阻断处理能通过抑制果实NOX活性来减少H2O2积累，促进了病害扩展。3.100mmol/L硅酸钠浸泡处理诱导了甜瓜的H2O2积累；处理果实12至48h后损伤接种Trichothecium roseum，果实的发病率和病斑面积显著降低。硅酸钠结合DPI处理时发现，DPI处理抑制了硅酸钠对甜瓜果实H2O2迸发的诱导，从而减弱了硅酸钠处理对果实粉霉病抗性的诱导效果。4.采用2-DE蛋白质组学技术，研究了硅酸钠处理及T. roseum挑战接种对甜瓜果实蛋白质组的影响，结果表明，在硅处理后48h内，共鉴定出81个差异表达蛋白，质谱鉴定和生物信息分析结果发现，这些蛋白属于能量、代谢、氧化还原稳态、病害与防卫反应、次生代谢、蛋白质折叠加工、细胞结构、转运、蛋白质合成以及未知功能等十大类。其中，8种蛋白(peroxidase,peroxidaseprecursor,heat shock protein70, hsp90-2-like, desiccation-related protein, stress-induced proteinsti1-like protein,26kDa phloem lectin和17kDa phloem lectin)与植物的防卫反应相关，直接或间接参与植物抗病性。5种蛋白（ascorbate oxidase, catalase,monodehydroascorbate reductase, glutathione transferase, glutathione s-transferase)属于抗氧化蛋白，涉及活性氧代谢。同时，一些能量代谢相关蛋白的调节也可能参与果实的抗病反应。5.采用iTRAQ技术分析了硅酸钠以及T. roseum挑战接种对甜瓜果实蛋白质组的影响，结果发现处理后36小时，未接种和接种组的处理诱导了94种蛋白的差异表达，这些蛋白涉及能量途径、蛋白质合成、防卫反应、代谢、信号传导、转录和其它等七种功能，其中一些防卫相关蛋白如hypersensitive-inducedresponseprotein, netting associated peroxidase, peroxidase， endochitinase MCHT-2,Thermoinhibition-associated THB-4protein, pollen coat protein, classical AGP和wound-induced protein等被鉴定上调表达，参与了果实的抗病反应。综上所述，硅酸钠具有诱导梨、苹果和甜瓜三种果实抗病性的能力，诱导的抗病性应答与过氧化氢积累、防卫相关酶活性提高以及蛋白质组表达调节密切相关。研究结果对进一步深入揭示硅酸钠诱导果实抗病性应答机理具有一定理论指导意义，对有效降低果实采后病害的发生和减少化学杀菌剂在采后病害控制中的使用也提供了重要的参考价值。更多还原

【Abstract】 Disease caused by fungal infection is a critical factor which is responsible forpostharvest loss and decay of fresh fruits and vegetables. Currently, chemicalfungicides treatment is primary and effective method for controlling these diseases.However, due to problems related to fungicide residues, environmental pollutions andfungicide resistance by pathogens, there is a worldwide trend to reduce or restrict touse of chemical fungicides. Induce resistance, as one kind of new and safe strategy forcontrolling postharvest disease, have a potential ability to instead or reduceapplication of chemical fungicides. Furthermore, previous studies have shown thattreatment with soluble silicate could reduce postharvest disease of some fruits throughinducing resistance. In the present study, fruits of ‘Zaosu’ pear,‘Fuji’ apple and‘Yujinxiang’ muskmelon were chosen as materials, and multiple approaches involvingthe postharvest pathology, biochemistry and proteomics have been used to investigatecontrol effective of sodium silicate treatment on major postharvest disease in the threekinds of fruits above mentioned, to study the effect of treatment on metabolism oraccumulation of hydrogen peroxide in fruits, and to analyze effect of treatment onproteomic changes in muskmelon fruit. Main results were following:1.100mM sodium silicate treatment can significantly inhibit blue mold caused byPenicillium expansum in ‘Zaosu’ pear fruit during storage. Moreover, enhancement ofperoxidase (POD) activity and reduction of activities of catalase (CAT) and ascorbateperoxidase (APX) were found in Si-treated fruit.0.1mmol/L sodium silicate treatmentby using inoculation method can also significantly control blue mold of ‘Zaosu’ pearfruit. However, Si treatment combined with DPI reduced the ability of controllingblue mold in fruit, which were consisted with inhibition of hydrogen peroxide (H2O2)content and NADPH oxidase (NOX) activity in integration treated fruit.2.200mM sodium silicate treatment resulted in alleviation of incidence of disease,and inhibition of development of lesion of apple fruit inoculated with P. expansum.Compared with the control, both superoxide dismutase (SOD) and NOX activities inthe treated fruit were significantly higher after treatment, while CAT and APXactivities were significantly lower，which were associated with the significant increaseof superoxide (02-) production and H202content after treatment respectively. Furthermore, the inhibition of the activity of NOX by DPI also resulted in inhibitionof H2O2content, and accelerating development of lesion.3. The timing of accumulation of H2O2and response to resistance againstTrichothecium roseum in melon fruit treated with100mM sodium silicate byimmersion method were investigated in this study. The results of dying by DABsuggested that the initiation of H2O2accumulation was4hours after treatment. From12to48hours after treatment, Si-treated fruit had ability to inhibiting pink rot.Meanwhile, the results of Si treatment combined with DPT indicated thataccumulation of H2O2in fruits at the time after24hours treatment were correlatedwith the ability to resistance against pink rot.4. To obtain unique insights regarding the effect of induced resistance onpostharvest Si treatments in muskmelons (cv. Yujinxiang) challenged with T. roseum,a complete proteome analysis was performed by using two dimensional gelselectrophoresis (2-DE) followed by matrix-assisted laser desorption ionization time offlight mass spectrometry (MALDI-TOF-MS). A total of81proteins were identified assignificantly up-or down-regulated in response to Si induction and T. roseuminoculation in fruits. After functional categorization, these proteins were attributed toenergy pathway, metabolism, redox homeostasis, disease/defense response, secondarymetabolism, protein metabolism, transporter and cell structure etc. Among them, both8defense response proteins including peroxidase, peroxidase precursor, heat shockprotein70, hsp90-2-like, desiccation-related protein, stress-induced protein sti1-likeprotein,26kDa phloem lectin and17kDa phloem lectin were correlated with defenceresponse, which were involved resistance directly or inderectly. Futhermore,5redoxhomeostasis associated proteins containing ascorbate oxidase, catalase,monodehydroascorbate reductase, glutathione transferase, glutathione s-transferasewere belongs to antiodixant proteins, which were involved to metabolism of reactiveoxygen species. In addition, more than10proteins identified as the enzymesassociated with energy metabolism were also involved in induced resistance ofmuskmelon fruits by Si and challenged by T. roseum.5. The effect of postharvest Si treatments on changes of proteome in muskmelonfruit at36hours after treatment were also investigated by iTRAQ technique. A total of 94polypeptides whose abundance changed in response to the elicitor were identified36hours after the treatments. These altered proteins attribute to energy pathway,protein synthesis, defence response, metabolism, signal transduction, transcription andothers. Among them, many proteins associtated with defence response such ashypersensitive-induced response protein, netting associated peroxidase, peroxidase，endochitinase MCHT-2, thermoinhibition-associated THB-4protein, pollen coatprotein, classical AGP and wound-induced protein were identified as up-regulatedproteins, which can involve in resistance response in fruits.In conclusion, the results of the present study indicated that sodium silicate hasan ability of inducing disease resistance in three fruits including pear, apple andmuskmelon. Meanwhile, the induced resistance of fruits in response to sodium silicateclearly correlated with the accumulation of hydrogen peroxide, enhancement ofactivities of enzymes related defense response, and regulation of proteomics in fruits.Furthermore, these results have an important significance of theoretical guidance forexploring the mechanisms of induced resistance of fruits in response to sodiumsilicate in the future. Moreover, this suggested sodium silicate could be a promisingstrategy in reducing postharvest disease and partially substituting chemical fungicidesto control postharvest disease in fruits and vegetables.更多还原