番茄SlAV2基因的克

发布时间：2018-03-03 11:03

本文选题：番茄　切入点：SlAN2　出处：《山东农业大学》2015年博士论文　论文类型：学位论文

【摘要】：花青素是类黄酮途径的产物之一,近年来受到越来越多的关注。花青素的积累受一系列环境和发育信号的诱导。花青素是一种抗氧化物质和光保护屏障,同时它在光保护、抗逆、授粉以及种子传播等许多生理过程中都发挥着重要的作用。另外,人类膳食中的花青素对健康也十分有益。花青素合成的调控主要发生在转录水平。花青素合成相关的结构基因受转录复合体激活,这些复合体由R2R3-MYB转录因子、bHLH转录因子和WD重复蛋白组成。SlAN2编码一个R2R3-MYB转录因子,调控番茄花青素的合成。植物遭受非生物胁迫时,体内代谢失衡会导致活性氧(ROS)大量积累。当胁迫加剧时,过量积累的ROS会对植物造成一系列的损伤。幸运的是,植物进化出了许多适应机制来对抗ROS的伤害,这些机制包括酶促和非酶促抗氧化防御体系。类胡萝卜素,抗坏血酸,谷胱甘肽以及类黄酮等都属于非酶促的抗氧化物质,它们可以清除ROS从而保护植物免受损害。花青素就是一种抗氧化物质,它的抗氧化性能主要归功于糖基上的羟基。番茄是研究果实成熟的模式植物,然而SlAN2与果实成熟的关系还不清楚。本研究从番茄中克隆得到SlAN2基因,并对该基因的表达和功能进行分析,主要结果如下:(1)分离得到SlAN2基因,包含一个828个碱基的开放阅读框,能编码275个氨基酸。(2)将SlAN2-GFP融合蛋白在洋葱表皮中瞬时表达,观察到该融合蛋白定位在细胞核中。(3)实时定量PCR(qRT-PCR)表明SlAN2的转录水平在茎中最高,在叶、花和根中水平适中,在果实中最低。SlAN2的表达水平受低温以及甲基紫晶(MV)的诱导上调。(4)构建正、反义表达载体,利用农杆菌进行番茄和烟草的遗传转化,获得正、反义转基因番茄植株和正义转基因烟草植株。对有卡那抗性的转基因番茄和烟草植株进行qRT-PCR检测,结果证明成功地获得了正、反义转基因番茄植株和正义转基因烟草植株。正义转基因番茄叶片的花青素含量升高,为野生型番茄的2.2-3.2倍,过表达SlAN2的烟草叶片花青素含量升高了100-284倍。(5)与野生型烟草相比,转基因烟草通过维持更高的叶绿素含量,净光合速率和光系统II的最大光化学效率来抵御低温胁迫。另外,在遭受低温胁迫时,转基因烟草的离子渗透更少,ROS水平更低,非酶促抗氧化性能更高。转基因烟草对由MV诱发的氧化胁迫的抗性也更强。与野生型烟草相比,转基因烟草在遭受氧化胁迫时,叶绿素含量降低的更少,离子渗透更少,能维持更高的光系统II的最大光化学效率。这些结果表明,SlAN2在烟草中的过量表达,使得转基因烟草中花青素大量积累,增强了转基因烟草对低温和氧化胁迫的抗性。(6)与野生型番茄相比,转基因番茄在抵御高温胁迫时,能够维持更高的鲜重、净光合速率和光系统II的最大光化学效率。另外,与野生型番茄相比,转基因番茄在遭受高温胁迫时,有更强的非酶促抗氧化性能,能维持更低的ROS水平和更高的PSII反应中心D1蛋白含量。这些结果说明,SlAN2在抵御高温胁迫时发挥着重要作用。(7)过表达SlAN2的番茄果实成熟后呈橙色,有软化快,乙烯释放量多等特点。SlAN2的过量表达,通过对类胡萝卜素合成相关基因的影响使类胡萝卜素积累减少;通过上调乙烯合成基因的表达,使乙烯释放量增加;通过降低果实硬度,使果实软化提前。另外在转基因番茄果实中,一个重要的果实成熟相关的基因,SlRIN被上调表达。以上结果说明SlAN2是一个重要的番茄果实成熟调控因子。
[Abstract]:Anthocyanin is one of the products of the flavonoid pathway, has attracted more and more attention in recent years. Induced anthocyanin accumulation by a series of environmental and developmental signals. Anthocyanin is an antioxidant and light protection barrier, while it is in the light protection, resistance, many physiological processes in pollination and seed dispersal plays an important role. In addition, human dietary anthocyanins is also very good for health. Anthocyanin synthesis regulation occurs mainly at the transcriptional level. The structure of genes related to anthocyanin biosynthesis transcription complex activation of these complexes by R2R3-MYB transcription factor, bHLH transcription factor and WD repeat protein.SlAN2 encoding a R2R3-MYB transcription factor, regulating anthocyanin synthesis in tomato. Plants are exposed to abiotic stress, metabolic imbalance will lead to the accumulation of reactive oxygen species (ROS). When the stress increased, excessive accumulation of ROS in plants By a series of injuries. Fortunately, plants have evolved many adaptation mechanism against ROS damage, these mechanisms include enzymatic and non enzymatic antioxidant defense system. Carotenoids, ascorbic acid, glutathione and flavonoids belong to non enzymatic antioxidants, they can remove ROS and protect plants against damage anthocyanin is a kind of antioxidant. The antioxidant properties, it is mainly attributed to the sugar hydroxyl groups on tomato is a model plant. Studies on fruit maturity, however, the relationship between SlAN2 and the fruit is not clear. This study cloned from tomato SlAN2 gene, and the expression and function of the genes were analyzed, mainly the results are as follows: (1) isolated SlAN2 gene contains a 828 BP open reading frame encoding 275 amino acid residues. (2) the fusion protein of SlAN2-GFP transient expression in onion epidermal cells To observe the fusion protein was localized in the nucleus. (3) real time quantitative PCR (qRT-PCR) showed that the transcriptional level of SlAN2 in the highest in leaves, stems, flowers and roots in the moderate level, and by low expression levels in the fruit of methyl Amethyst minimum.SlAN2 (MV) on the induction of the tune. (4) is constructed, antisense expression vector, tomato and tobacco by Agrobacterium mediated genetic transformation, positive, antisense transgenic tomato plants and transgenic tobacco plants. The justice of kanamycin resistant transgenic tomato and tobacco plants were detected by qRT-PCR, the results prove successful, antisense transgenic tomato plants and justice the transgenic tobacco plants. The transgenic tomato leaves increased anthocyanin content, 2.2-3.2 times of wild type tomato, expression of anthocyanin content in tobacco leaves of SlAN2 increased by 100-284 times. (5) compared with the wild type tobacco, transgenic tobacco by To maintain higher chlorophyll content, net photosynthetic rate and maximum photochemical efficiency of photosystem II to resist low temperature stress. In addition, in the low temperature stress, ion permeability of transgenic tobacco less, lower ROS levels, non enzymatic antioxidant performance higher. Transgenic tobacco resistance to MV induced by oxidative stress is also stronger. Compared with the wild type tobacco, transgenic tobacco caused by oxidative stress, the chlorophyll content decreased less, ion permeability less, can maintain the maximum photochemical efficiency of photosystem II higher. These results indicate that overexpression of SlAN2 in tobacco, the transgenic tobacco anthocyanin accumulation, enhanced resistance of transgenic tobacco low temperature stress and oxidation. (6) compared with the wild type tomato, transgenic tomato to resist high temperature stress, can maintain a higher fresh weight, net photosynthetic rate and the maximum optical system II The chemical efficiency. In addition, compared with the wild type tomato, transgenic tomato under high temperature stress, have a stronger non enzymatic antioxidant properties, can maintain lower ROS levels and higher PSII reaction center D1 protein content. These results suggest that SlAN2 in resisting high temperature plays an important role. (7) overexpression of SlAN2 in tomato fruit was ripe orange, softening quickly, overexpression of ethylene and.SlAN2 characteristics, through the influence of carotenoid biosynthetic genes to reduce the accumulation of carotenoids; through the expression of a synthetic base for the ethylene ethylene increases, increased release; by reducing the fruit hardness, the early fruit softening in transgenic tomato fruits. In addition, one of the most important fruit ripening related genes, SlRIN was up-regulated. These results suggest that SlAN2 is an important factor in regulating the ripening of tomato fruit.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q943.2

【共引文献】