【摘要】：柑橘果實(shí)從采收到消費(fèi)需要經(jīng)歷采后處理、貯藏、運(yùn)輸和銷售等環(huán)節(jié)。在這個(gè)過(guò)程中,柑橘果實(shí)容易受到一系列生物和非生物脅迫,這些脅迫能引起果實(shí)自身生理生化的變化,并最終導(dǎo)致果實(shí)風(fēng)味品質(zhì)下降,營(yíng)養(yǎng)物質(zhì)損失,失水及腐爛變質(zhì)。以青霉病、綠霉病和酸腐病為代表的侵染性病害是造成柑橘采后損失的主要原因。這些病害常常發(fā)生在采后果實(shí)抗性較弱的時(shí)候,并且多數(shù)病原菌能通過(guò)果實(shí)表皮的傷口、皮孔或破壞果皮組織的角質(zhì)層侵入果實(shí)內(nèi)部。目前,對(duì)柑橘采后侵染性病害的控制主要依賴于化學(xué)殺菌劑的使用,如抑霉唑,噻菌靈,嘧霉胺,咪鮮胺和咯菌腈等。然而,化學(xué)殺菌劑存在增強(qiáng)病原菌耐藥性以及危害環(huán)境和人體健康等問(wèn)題,所以人們一直尋找能夠代替化學(xué)殺菌劑的病害控制技術(shù)。利用生物的、化學(xué)的和物理的激發(fā)子誘導(dǎo)果實(shí)自身的抗病能力來(lái)防治采后病害,被認(rèn)為是一種安全無(wú)污染的病害防治新方法。這些激發(fā)子能誘導(dǎo)果實(shí)局部抗病性和系統(tǒng)抗病性,增強(qiáng)果實(shí)自身的非特異性抗性來(lái)抵御病原菌的侵襲。其中,水楊酸、膜醭畢赤酵母和殼寡糖作為三種典型的果實(shí)抗性激發(fā)子,能誘導(dǎo)多種果實(shí)自身抗性,增強(qiáng)果實(shí)抵御采后生物脅迫的能力。因此,本論文以柑橘果實(shí)為試材,利用這三種典型的外源激發(fā)子為處理手段,探討這三種激發(fā)子對(duì)柑橘采后病害的控制效力,對(duì)比它們誘導(dǎo)柑橘果實(shí)抗性能力的強(qiáng)弱。在此基礎(chǔ)上,借助轉(zhuǎn)錄組學(xué)和蛋白質(zhì)組學(xué)技術(shù),結(jié)合傳統(tǒng)生理生化分析方法,全面而系統(tǒng)的分析水楊酸、膜醭畢赤酵母和殼寡糖處理對(duì)柑橘果實(shí)基因表達(dá)、蛋白表達(dá)和物質(zhì)代謝的影響,以此探討激發(fā)子處理后柑橘果實(shí)抗性反應(yīng)的生物學(xué)機(jī)制。全文主要結(jié)果如下:(1)在損傷接種的模式下,采用2.5 mmol·L-1水楊酸,1×108 CFU·mL-1膜醭畢赤酵母細(xì)胞懸浮液和1.5%殼寡糖處理柑橘果實(shí),能顯著降低果實(shí)采后青霉病、綠霉病、酸腐病的發(fā)病率和病斑直徑,但三種激發(fā)子的控病能力因接種方式的不同而有所差異。在處理液與病原菌同孔接種的情況下,酵母對(duì)病害的控病能力最強(qiáng),其次是殼寡糖,水楊酸的效果最弱;在處理液與病原菌異孔接種的情況下,酵母與殼寡糖的控病能力相當(dāng),均強(qiáng)于水楊酸。(2)采用2.5mmol·l-1水楊酸,1×108cfu·ml-1膜醭畢赤酵母細(xì)胞懸浮液和1.5%殼寡糖浸泡柑橘果實(shí),能顯著降低柑橘果實(shí)貯藏期間自然發(fā)病率和病情指數(shù),且伴隨貯藏時(shí)間的延長(zhǎng),三者控病能力產(chǎn)生一定差異。在果實(shí)貯藏后期,殼寡糖的效果明顯好于膜醭畢赤酵母和水楊酸,酵母和水楊酸之間沒(méi)有顯著性差異。(3)利用rna-seq技術(shù),從水楊酸、膜醭畢赤酵母和殼寡糖處理的樣品中篩選出差異表達(dá)基因:水楊酸處理組2344個(gè),酵母處理組918個(gè),殼寡糖處理組1323個(gè)。熒光定量pcr驗(yàn)證實(shí)驗(yàn)結(jié)果表明rna-seq的數(shù)據(jù)真實(shí)可靠。這些差異基因主要參與代謝過(guò)程,細(xì)胞過(guò)程,單組織過(guò)程和刺激的響應(yīng)等生物學(xué)過(guò)程。差異基因代謝途徑分析結(jié)果表明,三種激發(fā)子刺激了柑橘果實(shí)次級(jí)代謝通路相關(guān)基因的轉(zhuǎn)錄表達(dá)。同時(shí),對(duì)碳代謝和氨基酸代謝相關(guān)通路基因的轉(zhuǎn)錄表達(dá)也有顯著影響。(4)利用itraq技術(shù),從水楊酸、膜醭畢赤酵母和殼寡糖處理的樣品中篩選出差異表達(dá)蛋白:水楊酸處理組109個(gè),酵母處理組327個(gè),殼寡糖處理組164個(gè)。這些差異蛋白主要參與代謝過(guò)程,細(xì)胞過(guò)程,單組織過(guò)程等生物學(xué)過(guò)程。代謝途徑分析結(jié)果表明,三種激發(fā)子能顯著影響柑橘果實(shí)碳代謝、次級(jí)代謝生物合成相關(guān)途徑和抗氧化相關(guān)代謝途徑;同時(shí),酵母和殼寡糖激子還能顯著影響柑橘果實(shí)逆境相關(guān)氨基酸代謝通路蛋白的表達(dá)水平。(5)柑橘果實(shí)經(jīng)水楊酸、膜醭畢赤酵母和殼寡糖處理后,對(duì)三種處理共有差異基因和共有差異蛋白的分析結(jié)果表明,苯丙烷類生物合成途徑在三種激發(fā)子誘導(dǎo)的柑橘果實(shí)抗性反應(yīng)中具有重要作用,且三種激發(fā)子對(duì)該通路上關(guān)鍵基因和關(guān)鍵蛋白表達(dá)模式的調(diào)控具有一致性。(6)利用氣相色譜-質(zhì)譜聯(lián)用儀(gc-ms)和氨基酸自動(dòng)分析儀,分析了水楊酸、膜醭畢赤酵母和殼寡糖處理后柑橘果皮主要初生代謝物—糖、有機(jī)酸和氨基酸的含量變化規(guī)律。結(jié)果顯示,三種激發(fā)子通過(guò)提高柑橘果皮中可溶性糖和三羧酸循環(huán)中關(guān)鍵的有機(jī)酸(檸檬酸、α-酮戊二酸、琥珀酸、蘋果酸和富馬酸)的含量,為抗病反應(yīng)提供能量和底物;通過(guò)累積滲透調(diào)節(jié)物質(zhì)和抗氧化相關(guān)的物質(zhì)(肌醇、脯氨酸、谷氨酸等),來(lái)增強(qiáng)細(xì)胞組織對(duì)滲透脅迫和氧化脅迫的耐受力,從而強(qiáng)化果實(shí)抗逆性;通過(guò)刺激在抗病反應(yīng)中與信號(hào)物質(zhì)和抗性物質(zhì)合成密切相關(guān)的初生代謝物(草酸、甲硫氨酸、苯丙氨酸)在果皮中的累積,直接間接的作用于果實(shí)的抗性反應(yīng)。(7)利用傳統(tǒng)的生理生化分析技術(shù)結(jié)合高效液相色譜(hplc),分析了水楊酸、膜醭畢赤酵母和殼寡糖處理對(duì)柑橘果實(shí)苯丙烷代謝途徑的影響。結(jié)果顯示,三種激發(fā)子能顯著增強(qiáng)果皮苯丙氨酸解氨酶(pal)、肉桂酸-4-羥基化酶(c4h)、4-香豆酸輔酶a連接酶(4cl)、過(guò)氧化物酶(pod)和多酚氧化酶(ppo)的活性,提高柑橘果皮中綠原酸、咖啡酸、阿魏酸和對(duì)香豆酸的含量,刺激木質(zhì)素的在果皮中的累積,進(jìn)而強(qiáng)化果實(shí)的結(jié)構(gòu)抗性和生化抗性。(8)結(jié)合轉(zhuǎn)錄組、蛋白組和常規(guī)生理生化分析的研究結(jié)果,從轉(zhuǎn)錄水平、翻譯水平和產(chǎn)物水平,全面證實(shí)了苯丙烷代謝通路參與了柑橘果實(shí)對(duì)水楊酸、膜醭畢赤酵母和殼寡糖誘導(dǎo)的抗病性應(yīng)答過(guò)程,說(shuō)明激活苯丙烷代謝途徑關(guān)鍵酶的轉(zhuǎn)錄表達(dá),誘導(dǎo)該條途徑上抗性物質(zhì)的合成是這三種激發(fā)子誘導(dǎo)柑橘果實(shí)抗病性的共有機(jī)制。
[Abstract]:Citrus fruit from harvest to consumption needs to undergo post harvest processing, storage, transportation and sales. In this process, citrus fruit is vulnerable to a series of biological and abiotic stresses, which can cause physiological and biochemical changes of the fruit itself, and eventually lead to the decline of fruit flavor quality, loss of nutrients, loss of water and decay. Infective diseases represented by Penicillium, green mildew and acid rot are the main causes of Postharvest loss of citrus. These diseases often occur when the resistance is weak in postharvest fruits, and most of the pathogens can invade the fruit through the wound of the fruit epidermis, skin holes or the stratum corneum that destroy the skin tissue. The control of infective diseases mainly depends on the use of chemical fungicides, such as anti mycophenazole, thiamethazine, pyrimethamine, imidamine and nitrile. However, chemical fungicides have been found to enhance the drug resistance of pathogens and harm the environment and human health. So people have been looking for disease control techniques that can replace chemical fungicides. Material, chemical and physical elicitors induce the disease resistance of fruit to control postharvest diseases. It is considered a new and safe and non polluting disease control method. These elicitors can induce local disease resistance and systemic disease resistance of fruit, enhance the non specific resistance of fruit itself to resist the invasion of pathogenic bacteria. As three typical fruit resistant excitations, Pichia pastoris and chitosan oligosaccharides can induce a variety of fruit self resistance and enhance the ability to resist postharvest biological stress. Therefore, this paper uses citrus fruit as a test material and uses these three typical exogenous excite as the processing hand to discuss the three kinds of elicitors on the postharvest diseases of Citrus On this basis, the effects of salicylic acid, Pichia pastoris and oligosaccharides on citrus fruit gene expression, protein expression and substance metabolism were systematically and systematically analyzed with the help of transcriptional and proteomics techniques combined with traditional physiological and biochemical methods. The main results are as follows: (1) under the mode of inoculation, 2.5 mmol. L-1 salicylic acid, 1 x 108 CFU / mL-1 membrane and 1.5% chitosan oligosaccharides can be used to treat citrus fruit, which can significantly reduce the Postharvest Penicillium, green mould and acid rot The incidence of disease and the diameter of the disease spot, but the control ability of the three kinds of elicitors varies according to the different inoculation methods. In the case of the inoculation of the treatment solution and the pathogen in the same hole, the yeast has the strongest control ability to the disease, followed by the chitosan oligosaccharide, the salicylic acid is the weakest; the yeast and the chitosan oligosaccharides are in the condition of the inoculation of the treatment solution and the pathogen. The ability of controlling disease was equal to that of salicylic acid. (2) using 2.5mmol L-1 salicylic acid, 1 x 108cfu / ml-1 membrane and 1.5% shell oligosaccharides soaked citrus fruit, can significantly reduce the natural incidence and disease index of citrus fruit during storage, and with the prolongation of storage time, the disease control ability of the three people is different. After storage, the effect of chitosan oligosaccharides was better than that of Pichia pastoris and salicylic acid, and there was no significant difference between yeast and salicylic acid. (3) the differential expression genes were screened from salicylic acid, Pichia pastoris and chitosan oligosaccharides by RNA-seq Technology: 2344 of salicylic acid treatment group, 918 in yeast treatment group and 1 in chitosan oligosaccharide treatment group. 323. The results of fluorescence quantitative PCR verification showed that the data of RNA-seq were true and reliable. These differential genes were mainly involved in biological processes such as metabolic process, cell process, single tissue process and response to stimulation. The results of differential gene metabolism pathway analysis showed that three kinds of elicitors stimulated the transfer of related genes in the secondary metabolism pathway of citrus fruit. (4) the differential expression proteins were screened from salicylic acid, Pichia pastoris and chitosan oligosaccharides by iTRAQ technology, 109 of salicylic acid treatment group, 327 in yeast treatment group and 164 in chitosan oligosaccharide treatment group. Mainly involved in biological processes such as metabolic processes, cell processes, and single tissue processes. Metabolic pathways analysis showed that the three excites could significantly affect the carbon metabolism of citrus fruit, secondary metabolism biosynthesis pathway and antioxidant related metabolic pathway, while yeast and chitosan oligosaccharide can significantly affect the citrus fruit adversity related ammonia (5) after treatment of citrus fruits with salicylic acid, Pichia pastoris and chitosan oligosaccharides, the analysis of three different genes and common differential proteins showed that the phenylpropanoid biosynthesis pathway played an important role in the resistance response of the citrus fruit induced by three elicitors and three excitations. The regulation of the key genes and key protein expression patterns on the pathway was consistent. (6) the main primary metabolites of salicylic acid, Pichia pastoris and oligosaccharides were analyzed by using gas chromatography-mass spectrometry (GC-MS) and amino acid analyzer. The changes of the contents of main primary metabolites, organic acids and amino acids in Citrus peel after treatment were analyzed. The results showed that three kinds of elicitors could provide energy and substrate for the resistance to disease by increasing the content of the key organic acids (citric acid, alpha ketopamyl diacid, succinic acid, malic acid and fumaric acid) in the citrus fruit peel, which were the key organic acids (citric acid, alpha ketopamyl diacid, succinic acid, malic acid and fumaric acid), and through the accumulation of osmotic substances and antioxidative related substances (inositol, proline, glutamic acid, etc.) In order to enhance the tolerance of cell tissue to osmotic and oxidative stress, and thus strengthen the resistance to fruit, the accumulation of primary metabolites (oxalic acid, methionine, phenylalanine) in the pericarp, which are closely related to the synthesis of signal and resistant substances in the resistance to disease, directly indirectly acts on the resistance reaction of the fruit. (7) The effects of salicylic acid, Pichia pastoris and chitosan oligosaccharides on the metabolic pathway of benzopropane in citrus fruits were analyzed with traditional physiological and biochemical analysis technique combined with high performance liquid chromatography (HPLC). The results showed that three excites could significantly enhance the pericarp phenylalanine ammonia lyase (PAL), cinnamic acid -4- hydroxylase (C4H), 4- coumaric acid coenzyme a connection The activity of enzyme (4CL), peroxidase (POD) and polyphenol oxidase (PPO), increase the content of Lv Yuan acid, caffeic acid, ferulic acid and coumaric acid in citrus peel, stimulate the accumulation of lignin in the skin, and then strengthen the structural resistance and Biochemical Resistance of the fruit. (8) combined with the results of transcriptional, protein and routine physiological and biochemical analysis, From the transcriptional level, translation level and product level, the phenylpropane metabolic pathway involved in the response of citrus fruit to salicylic acid, Pichia pastoris and chitosan oligosaccharides, indicating the transcriptional expression of the key enzymes activating the phenylpropane metabolism pathway, and the induction of the synthesis of the resistant substances on the route is the three elicitors. The common mechanism of citrus fruit resistance to disease.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S436.66;TS255.3
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本文編號(hào)：2161911