转基因聚合影响番茄抗坏血酸含量的研究

发布时间：2018-05-14 16:47

  本文选题：番茄 + 抗坏血酸　； 参考：《华中农业大学》2017年硕士论文

【摘要】：抗坏血酸(L-ascorbic acid,AsA)是动物和植物都必须的重要代谢物之一。在植物中它是能够清除活性氧的抗氧化物,参与很多代谢过程。在人体中,AsA可以清除体内各种代谢反应产生的自由基,是人类不可缺少的营养物质之一,然而人类进化过程中丧失了合成AsA的能力,必须通过食物摄取体内所需的AsA。番茄(Solanum lycopersicum)是植物学果实研究的重要的模式植物,且含有丰富的抗坏血酸,因此,番茄抗坏血酸的研究在营养品质领域中有着极其重要的意义,也成为了科研者研究的重点。目前,AsA合成的几条途径已经研究较为清晰,途径中的关键基因分别被克隆并鉴定了其功能。植物中大部分性状都是由多基因决定并调控的,其中AsA含量也是多基因决定的。之前有报道将多个基因聚合提高了植物的耐盐性、抗病性以及氨基酸的含量等,但是,还没有基因聚合在抗坏血酸方面应用的研究。本课题是将实验室已保存的AsA途径中关键基因的超量转基因系进行聚合杂交,通过生理生化实验来研究转基因聚合对番茄中AsA含量的影响。本课题的主要结果如下:1、将实验室保存的GMP、GME、GGP、GPP、GalUR、MIOX、MDHAR和DHAR超量转基因植株聚合杂交得到二价聚合植株GMP×GME、GGP×GPP、GalUR×MIOX、MDHAR×DHAR和四价聚合植株GMP×GME×GGP×GPP,六价聚合植株GMP×GME×GGP×GPP×MDHAR×DHAR,并且通过阳性检测,筛选到具有目的基因的植株。2、应用TCA抽提AsA的方法,测定了对照Ailsa Criag(AC)与单价转基因以及聚合植株的抗坏血酸含量,结果表明,在叶片中聚合植株与AC相比,AsA含量显著性增加,而在果实中除GGP×GPP植株没有显著性变化,其他聚合植株的AsA积累均显著性增加。应用qPCR对聚合植株中AsA合成代谢相关基因的表达量进行分析,发现除GMP×GME×GGP×GPP的叶片中AsA的合成基因和代谢基因受到抑制外其他聚合植株在叶片和果实中的合成代谢基因均呈上调表达,说明了聚合杂交导致多基因多点调控AsA,最终增加了AsA的含量。3、通过对在16h光照/8h黑暗的条件下生长的一个月苗龄的植株叶片进行测定AsA含量来分析聚合植株在光诱导下的规律,结果说明,聚合植株对光更加敏感,使聚合植株在此期间内的波动振幅变大,但是AsA含量随昼夜变化的趋势是没有改变。同时,通过AsA合成前体的饲喂试验发现,由于在叶片和果实中植株通过不同的合成途径合成AsA,在叶片中,主要是D-甘露糖/L-半乳糖途径合成AsA,而在果实中聚合植株GMP×GME、GMP×GME×GGP×GPP在果实中三条途径都同时发挥作用合成AsA,使得聚合植株的AsA的含量增加,GGP×GPP聚合植株则只是由D-甘露糖/L-半乳糖途径合成AsA,导致AsA含量没有显著性变化。4、通过对AsA合成途径中关键基因酶GMP、GalUR酶的活性测定,发现聚合植株中这两个酶都显著性的增加,说明转基因聚合增强了关键合成酶的酶活力,从而提高了AsA的积累。同时,通过甲基紫精模拟氧化胁迫,对叶绿素、丙二醛以及H2O2的检测说明,聚合植株也增强了对氧化逆境的抗性。5、为了探究聚合植株中源与库的关系,我们通过对叶柄和果柄以及其卸载液的AsA含量测定,聚合植株与对照AC相比果柄中的AsA含量显著性增加,结合聚合植株绿熟期的果实饲喂AsA与AgNO3组织定位的结果显示,聚合植株相比对照增强了AsA的转运能力。同时,聚合植株也改变了植株很多农艺性状,果实的形状和重量,茎粗,以及可溶性固形物等等。
[Abstract]:L-ascorbic acid (AsA) is one of the important metabolites of animals and plants. In plants, it is an antioxidant that can scavenge active oxygen and participates in many metabolic processes. In human body, AsA can remove free radicals produced by various metabolic reactions in the body. It is one of the essential nutrients in human beings. However, human evolution has evolved. In the process of losing the ability to synthesize AsA, the AsA. tomato (Solanum lycopersicum) required by food intake is an important model plant in the study of Botany fruit, and it contains abundant ascorbic acid. Therefore, the study of tomato ascorbic acid has a very important significance in the field of nutritional quality, and it has also become a research researcher. At present, several approaches to AsA synthesis have been studied clearly. The key genes in the pathway are cloned and identified respectively. Most of the traits in plants are determined and regulated by multiple genes, in which the AsA content is also determined by multiple genes. However, there is no study on the application of gene polymerization in ascorbic acid. This topic is to study the effect of transgenic polymerization on the content of AsA in tomato by physiological and biochemical experiments. The main conclusion of this topic is to study the effect of the overtransgenic lines of the key genes in the AsA pathway preserved in the laboratory. The results are as follows: 1, GMP, GME, GGP, GPP, GalUR, MIOX, MDHAR and DHAR were polymerized to get the two valence polymerized plant GMP x GME, GGP x GPP, and the tetravalent polymerized plant. .2, a plant with a target gene, was used to extract AsA by TCA. The content of ascorbic acid in the control Ailsa Criag (AC) and the monovalent transgenic plants and the polymerized plants was measured. The results showed that the content of AsA in the leaves was significantly higher than that of AC, but there was no significant change in the GGP x GPP plants in the fruit and the AsA product of the other polymerized plants. The expression of AsA anabolism related genes in the polymerized plants was analyzed with qPCR. It was found that the synthetic and metabolic genes of the other polymerized plants in the leaves and fruits were up to be up-regulated in the leaves and fruits of the plants except the GMP x GME x GGP x GPP leaves and the inhibition of the gene and metabolic genes of AsA. AsA was regulated by multiple genes, and the content of AsA was finally increased by.3. The results showed that the polymerization plants were more sensitive to light by measuring the content of AsA in the leaves of the one month seedling of the one month seedling growing in 16h light /8h dark conditions, which showed that the fluctuating amplitude of the polymerized plants increased in this period. It is the trend that the AsA content changes with the day and night. At the same time, the feeding test of the AsA synthesis precursor found that the plants were synthesized by different synthetic routes in the leaves and fruits. In the leaves, the mainly D- mannose /L- galactose pathway synthesized AsA, and the GMP x GME of the plant was polymerized in the fruit, GMP * GME x GGP * GPP was in the fruit. The three ways both play a role in the synthesis of AsA, which makes the content of AsA in the polymerized plants increase. The GGP x GPP polymerized plants are only synthesized by D- mannose /L- galactose pathway to AsA, resulting in no significant change in the AsA content.4. Through the assay of the key gene enzyme GMP, the activity determination of the GalUR enzyme, the two enzymes in the polymerized plants are found. The significant increase indicated that the transgenic polymerization enhanced the enzyme activity of the key synthase and increased the accumulation of AsA. At the same time, the detection of chlorophyll, malondialdehyde and H2O2 through the simulation of oxidative stress by methyl violet essence showed that the polymerized plants also enhanced the resistance to oxidative stress.5, in order to explore the relationship between the source and the library in the polymerized plants. Through the determination of the AsA content of the petiole, fruit handle and its unloading liquid, the AsA content in the stem was significantly increased compared with the control AC. The results showed that the aggregated plants enhanced the transport capacity of AsA compared with the control, and the polymerized plants also changed the plant of the AsA. There are many agronomic traits, fruit shape and weight, stem diameter, soluble solids and so on.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S641.2

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