發(fā)布時(shí)間：2018-05-14 16:47

  本文選題：番茄 + 抗壞血酸　； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：抗壞血酸(L-ascorbic acid,AsA)是動(dòng)物和植物都必須的重要代謝物之一。在植物中它是能夠清除活性氧的抗氧化物,參與很多代謝過程。在人體中,AsA可以清除體內(nèi)各種代謝反應(yīng)產(chǎn)生的自由基,是人類不可缺少的營養(yǎng)物質(zhì)之一,然而人類進(jìn)化過程中喪失了合成AsA的能力,必須通過食物攝取體內(nèi)所需的AsA。番茄(Solanum lycopersicum)是植物學(xué)果實(shí)研究的重要的模式植物,且含有豐富的抗壞血酸,因此,番茄抗壞血酸的研究在營養(yǎng)品質(zhì)領(lǐng)域中有著極其重要的意義,也成為了科研者研究的重點(diǎn)。目前,AsA合成的幾條途徑已經(jīng)研究較為清晰,途徑中的關(guān)鍵基因分別被克隆并鑒定了其功能。植物中大部分性狀都是由多基因決定并調(diào)控的,其中AsA含量也是多基因決定的。之前有報(bào)道將多個(gè)基因聚合提高了植物的耐鹽性、抗病性以及氨基酸的含量等,但是,還沒有基因聚合在抗壞血酸方面應(yīng)用的研究。本課題是將實(shí)驗(yàn)室已保存的AsA途徑中關(guān)鍵基因的超量轉(zhuǎn)基因系進(jìn)行聚合雜交,通過生理生化實(shí)驗(yàn)來研究轉(zhuǎn)基因聚合對番茄中AsA含量的影響。本課題的主要結(jié)果如下:1、將實(shí)驗(yàn)室保存的GMP、GME、GGP、GPP、GalUR、MIOX、MDHAR和DHAR超量轉(zhuǎn)基因植株聚合雜交得到二價(jià)聚合植株GMP×GME、GGP×GPP、GalUR×MIOX、MDHAR×DHAR和四價(jià)聚合植株GMP×GME×GGP×GPP,六價(jià)聚合植株GMP×GME×GGP×GPP×MDHAR×DHAR,并且通過陽性檢測,篩選到具有目的基因的植株。2、應(yīng)用TCA抽提AsA的方法,測定了對照Ailsa Criag(AC)與單價(jià)轉(zhuǎn)基因以及聚合植株的抗壞血酸含量,結(jié)果表明,在葉片中聚合植株與AC相比,AsA含量顯著性增加,而在果實(shí)中除GGP×GPP植株沒有顯著性變化,其他聚合植株的AsA積累均顯著性增加。應(yīng)用qPCR對聚合植株中AsA合成代謝相關(guān)基因的表達(dá)量進(jìn)行分析,發(fā)現(xiàn)除GMP×GME×GGP×GPP的葉片中AsA的合成基因和代謝基因受到抑制外其他聚合植株在葉片和果實(shí)中的合成代謝基因均呈上調(diào)表達(dá),說明了聚合雜交導(dǎo)致多基因多點(diǎn)調(diào)控AsA,最終增加了AsA的含量。3、通過對在16h光照/8h黑暗的條件下生長的一個(gè)月苗齡的植株葉片進(jìn)行測定AsA含量來分析聚合植株在光誘導(dǎo)下的規(guī)律,結(jié)果說明,聚合植株對光更加敏感,使聚合植株在此期間內(nèi)的波動(dòng)振幅變大,但是AsA含量隨晝夜變化的趨勢是沒有改變。同時(shí),通過AsA合成前體的飼喂試驗(yàn)發(fā)現(xiàn),由于在葉片和果實(shí)中植株通過不同的合成途徑合成AsA,在葉片中,主要是D-甘露糖/L-半乳糖途徑合成AsA,而在果實(shí)中聚合植株GMP×GME、GMP×GME×GGP×GPP在果實(shí)中三條途徑都同時(shí)發(fā)揮作用合成AsA,使得聚合植株的AsA的含量增加,GGP×GPP聚合植株則只是由D-甘露糖/L-半乳糖途徑合成AsA,導(dǎo)致AsA含量沒有顯著性變化。4、通過對AsA合成途徑中關(guān)鍵基因酶GMP、GalUR酶的活性測定,發(fā)現(xiàn)聚合植株中這兩個(gè)酶都顯著性的增加,說明轉(zhuǎn)基因聚合增強(qiáng)了關(guān)鍵合成酶的酶活力,從而提高了AsA的積累。同時(shí),通過甲基紫精模擬氧化脅迫,對葉綠素、丙二醛以及H2O2的檢測說明,聚合植株也增強(qiáng)了對氧化逆境的抗性。5、為了探究聚合植株中源與庫的關(guān)系,我們通過對葉柄和果柄以及其卸載液的AsA含量測定,聚合植株與對照AC相比果柄中的AsA含量顯著性增加,結(jié)合聚合植株綠熟期的果實(shí)飼喂AsA與AgNO3組織定位的結(jié)果顯示,聚合植株相比對照增強(qiáng)了AsA的轉(zhuǎn)運(yùn)能力。同時(shí),聚合植株也改變了植株很多農(nóng)藝性狀,果實(shí)的形狀和重量,莖粗,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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S641.2

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