水杨酸、膜醭毕赤酵母、壳寡糖诱导柑橘果实抗病性及其生物学机制研究
发布时间：2018-08-03 13:38
【摘要】：柑橘果实从采收到消费需要经历采后处理、贮藏、运输和销售等环节。在这个过程中,柑橘果实容易受到一系列生物和非生物胁迫,这些胁迫能引起果实自身生理生化的变化,并最终导致果实风味品质下降,营养物质损失,失水及腐烂变质。以青霉病、绿霉病和酸腐病为代表的侵染性病害是造成柑橘采后损失的主要原因。这些病害常常发生在采后果实抗性较弱的时候,并且多数病原菌能通过果实表皮的伤口、皮孔或破坏果皮组织的角质层侵入果实内部。目前,对柑橘采后侵染性病害的控制主要依赖于化学杀菌剂的使用,如抑霉唑,噻菌灵,嘧霉胺,咪鲜胺和咯菌腈等。然而,化学杀菌剂存在增强病原菌耐药性以及危害环境和人体健康等问题,所以人们一直寻找能够代替化学杀菌剂的病害控制技术。利用生物的、化学的和物理的激发子诱导果实自身的抗病能力来防治采后病害,被认为是一种安全无污染的病害防治新方法。这些激发子能诱导果实局部抗病性和系统抗病性,增强果实自身的非特异性抗性来抵御病原菌的侵袭。其中,水杨酸、膜醭毕赤酵母和壳寡糖作为三种典型的果实抗性激发子,能诱导多种果实自身抗性,增强果实抵御采后生物胁迫的能力。因此,本论文以柑橘果实为试材,利用这三种典型的外源激发子为处理手段,探讨这三种激发子对柑橘采后病害的控制效力,对比它们诱导柑橘果实抗性能力的强弱。在此基础上,借助转录组学和蛋白质组学技术,结合传统生理生化分析方法,全面而系统的分析水杨酸、膜醭毕赤酵母和壳寡糖处理对柑橘果实基因表达、蛋白表达和物质代谢的影响,以此探讨激发子处理后柑橘果实抗性反应的生物学机制。全文主要结果如下:(1)在损伤接种的模式下,采用2.5 mmol·L-1水杨酸,1×108 CFU·mL-1膜醭毕赤酵母细胞悬浮液和1.5%壳寡糖处理柑橘果实,能显著降低果实采后青霉病、绿霉病、酸腐病的发病率和病斑直径,但三种激发子的控病能力因接种方式的不同而有所差异。在处理液与病原菌同孔接种的情况下,酵母对病害的控病能力最强,其次是壳寡糖,水杨酸的效果最弱;在处理液与病原菌异孔接种的情况下,酵母与壳寡糖的控病能力相当,均强于水杨酸。(2)采用2.5mmol·l-1水杨酸,1×108cfu·ml-1膜醭毕赤酵母细胞悬浮液和1.5%壳寡糖浸泡柑橘果实,能显著降低柑橘果实贮藏期间自然发病率和病情指数,且伴随贮藏时间的延长,三者控病能力产生一定差异。在果实贮藏后期,壳寡糖的效果明显好于膜醭毕赤酵母和水杨酸,酵母和水杨酸之间没有显著性差异。(3)利用rna-seq技术,从水杨酸、膜醭毕赤酵母和壳寡糖处理的样品中筛选出差异表达基因:水杨酸处理组2344个,酵母处理组918个,壳寡糖处理组1323个。荧光定量pcr验证实验结果表明rna-seq的数据真实可靠。这些差异基因主要参与代谢过程,细胞过程,单组织过程和刺激的响应等生物学过程。差异基因代谢途径分析结果表明,三种激发子刺激了柑橘果实次级代谢通路相关基因的转录表达。同时,对碳代谢和氨基酸代谢相关通路基因的转录表达也有显著影响。(4)利用itraq技术,从水杨酸、膜醭毕赤酵母和壳寡糖处理的样品中筛选出差异表达蛋白:水杨酸处理组109个,酵母处理组327个,壳寡糖处理组164个。这些差异蛋白主要参与代谢过程,细胞过程,单组织过程等生物学过程。代谢途径分析结果表明,三种激发子能显著影响柑橘果实碳代谢、次级代谢生物合成相关途径和抗氧化相关代谢途径;同时,酵母和壳寡糖激子还能显著影响柑橘果实逆境相关氨基酸代谢通路蛋白的表达水平。(5)柑橘果实经水杨酸、膜醭毕赤酵母和壳寡糖处理后,对三种处理共有差异基因和共有差异蛋白的分析结果表明,苯丙烷类生物合成途径在三种激发子诱导的柑橘果实抗性反应中具有重要作用,且三种激发子对该通路上关键基因和关键蛋白表达模式的调控具有一致性。(6)利用气相色谱-质谱联用仪(gc-ms)和氨基酸自动分析仪,分析了水杨酸、膜醭毕赤酵母和壳寡糖处理后柑橘果皮主要初生代谢物—糖、有机酸和氨基酸的含量变化规律。结果显示,三种激发子通过提高柑橘果皮中可溶性糖和三羧酸循环中关键的有机酸(柠檬酸、α-酮戊二酸、琥珀酸、苹果酸和富马酸)的含量,为抗病反应提供能量和底物;通过累积渗透调节物质和抗氧化相关的物质(肌醇、脯氨酸、谷氨酸等),来增强细胞组织对渗透胁迫和氧化胁迫的耐受力,从而强化果实抗逆性;通过刺激在抗病反应中与信号物质和抗性物质合成密切相关的初生代谢物(草酸、甲硫氨酸、苯丙氨酸)在果皮中的累积,直接间接的作用于果实的抗性反应。(7)利用传统的生理生化分析技术结合高效液相色谱(hplc),分析了水杨酸、膜醭毕赤酵母和壳寡糖处理对柑橘果实苯丙烷代谢途径的影响。结果显示,三种激发子能显著增强果皮苯丙氨酸解氨酶(pal)、肉桂酸-4-羟基化酶(c4h)、4-香豆酸辅酶a连接酶(4cl)、过氧化物酶(pod)和多酚氧化酶(ppo)的活性,提高柑橘果皮中绿原酸、咖啡酸、阿魏酸和对香豆酸的含量,刺激木质素的在果皮中的累积,进而强化果实的结构抗性和生化抗性。(8)结合转录组、蛋白组和常规生理生化分析的研究结果,从转录水平、翻译水平和产物水平,全面证实了苯丙烷代谢通路参与了柑橘果实对水杨酸、膜醭毕赤酵母和壳寡糖诱导的抗病性应答过程,说明激活苯丙烷代谢途径关键酶的转录表达,诱导该条途径上抗性物质的合成是这三种激发子诱导柑橘果实抗病性的共有机制。
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.66;TS255.3
，
本文编号：2161911
[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.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S436.66;TS255.3
，
本文编号：2161911
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