【作者】 杨涛；

【导师】 张余洋；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2017， 硕士

【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。更多还原

【Abstract】 Ascorbic acid(L-ascorbate acid,AsA)is an important antioxidant that can remove reactive oxygen species in plants.It is also involved in a variety of metabolic processes in plants.In the human body,AsA can remove the free radicals in the body,and it is an indispensable nutrient,but the human lose the ability to synthesize AsA,so we have to absorb necessary ascorbic acid from fresh vegetables and fruits.Tomato(Solanum lycopersicum)is an important model plant in botanical research and it is rich in ascorbic acid.Therefore,the study of ascorbic acid content in tomato plays an important role in nutrient quality and has become the focus of the research.Now AsA synthesis pathway is elucidated,and the key genes of the pathways are also studied clearly.Most of the traits are controlled by multi-gene in plants,and AsA content is also controlled by ploygene.Previously it is reported that transgenic pyramiding improves the plant’s salt tolerance,disease resistance and amino acid content and so on,but there is no gene polymerization in ascorbic acid research,so in this study,the overexpression transgenic lines of the key genes of the As A pathway are studied by pollination and pyramiding,the effects of transgenic polymerization on AsA in tomato were analyzed by physiological and biochemical experiments.The main results of this topic are as follows:1.Hybridizing the transgenic lines overexpressing GMP,GME,GGP,GGP,GalUR,MIOX,MDHAR and DHAR by artificial pollination,then the GMP×GME,GGP×GPP,GalUR×MIOX,MDHAR×DHAR,GMP×GME×GGP ×GPP and GMP×GME×GGP×GPP×MDHAR×DHAR have been obtained,and the target genes of pyramiding lines were screened by positive detection.2.By using the method of TCA to measure AsA of Ailsa Criag(AC),single gene and pyramiding lines,the results show that compared with the AC,the As A content of leaf and fruit in pyramiding lines are significantly increased,except that the fruits of GGP×GPP line did not change significantly.The qPCR is used to analyze the relative expression of As A synthesis and metabolism related genes in the plant,and we found that except for the AsA metabolic and synthetic related genes in leaves of GMP×GME×GGP×GPP lines,gene expression in leaves and fruits of other pyramding lines were enhanced.It is indicated that the AsA content is controlled and regulated by multi-gene at multi-point.3.We measured the AsA content of one-month seedling at 16 h Light/8 h Dark,the result showed the light-induction on AsA in leaves of pyramiding lines only affected the AsA content and the fluctuation amplitude,and did not alter the original trend of AsA accumulation along circadian cycle.And by the results of AsA synthetic precursor feeding showed that gene pyramiding alter the AsA biosynthesis pathway contribution in leaves and fruits,and subsequently affect ascorbate content.It is assumed that the main cause of increasing AsA content was the contribution of D-mannose/L-galactose(D-Man/L-Gal)pathway in leaf,and for the GMP×GME,GMP×GME×GGP×GPP lines AsA content level was increased compared with wild type,for which three pathways were attributive to inceased ascorbate content,however,GGP×GPP lines mainly use the D-Man/L-Gal pathway.4.The enzyme activity of the AsA synthetic enzyme such as GMP,GalUR were measured,the result showed that the two enzymes in pyramiding lines were significantly increased,which is possibly the cause of increased AsA content.The methyl viologen simulated oxidation adversity,the chlorophyll,malondialdehyde and H2O2 were measured,and it showed that transgenic pyramiding also improved the resistance to oxidative stress.5.In oder to study the source and the sink regarding AsA,we measured the AsA content in fruit and leaf petioles and their exudates.We found that the AsA content of the fruit petiole and its exudate in the pyramiding lines was higher compared with AC,and combining the result of the AsA content after feeding AsA in the green ripe stage of AC and pyramiding lines,we suggested transgenic pyramiding enhanced the transportation capacity.In the addition,a lot of agronomic traits in the pyramiding lines were also altered,such as the shape and weight of the fruit,stem diameter,and the soluble solids and so on.更多还原