不结球白菜抗坏血酸合成代谢相关基因表达及BcGME功能分析

发布时间：2018-06-10 13:03

本文选题：不结球白菜 + 苏州青　；参考：《南京农业大学》2016年硕士论文

【摘要】：抗坏血酸(L-Ascorbic acid,AsA),又称维生素C,是动植物体中广泛存在的一种水溶性抗氧化剂(Gallie 2013)。植物中合成的AsA不仅能使无法合成AsA的人类及一些动物获得丰富的维生素C源,而且还能维持植物体的正常生长发育和代谢(安华明2004)。近年来广泛通过基因工程技术提高农作物的AsA含量,不仅有助于人体获得较高水平的维生素C,同时也对增强植株自身的抗性,提高农产品的营养品质具有重要影响。本文探究了胁迫和激素处理下不结球白菜抗坏血酸含量的变化与相关基因表达量之间的关联,为不结球白菜的优质育种奠定理论基础。本文主要研究结果如下:1.本研究旨在了解胁迫下不结球白菜抗坏血酸含量的变化,进一步找出调节抗坏血酸含量积累的关键基因。以'苏州青'为材料,分别进行了过氧化氢胁迫和水淹胁迫处理,测定了其AsA含量以及合成代谢相关基因的表达量。结果表明,两种处理下AsA含量均呈先上升后下降的趋势,过氧化氢胁迫下GME和GPP基因表达量的变化趋势与AsA含量变化趋势相一致,而水淹胁迫下PMM、GME和VTC2基因表达量的变化趋势与AsA含量变化趋势一致,说明GME基因可能是两种胁迫下均发挥作用的关健基因。2.以'苏州青'为材料,分别以0、10、50、100和200 μmol·L-1的脱落酸和乙烯处理,旨在了解激素处理下不结球白菜抗坏血酸的含量变化,进一步找出调节AsA含量变化的关键基因。结果显示:不同浓度ABA处理后AsA含量变化趋势不同,基因表达量分析发现较低浓度ABA(10 μmol·L-1,50μmol·L-1和100 μmol·L-1)处理组PMM基因表达量变化趋势与AsA含量变化趋势一致。乙烯处理后AsA含量呈先升高后下降趋势;10 μmol·L-1和50 μmol·L-1乙烯处理组GPP基因表达量变化趋势与AsA含量变化趋势一致,而100 μmol·L-1和200 μmol·L-1乙烯处理组PMM和GPP基因表达量变化趋势与AsA含量变化趋势一致。因此推测在脱落酸和乙烯处理下,PMM和GPP基因可能分别是其AsA生物合成途径的关键基因。3.以不结球白菜'苏州青'为材料,同源克隆了BcG E基因的全长,应用生物信息学分析了其氨基酸序列,并且构建了 BcGME-PCVA沉默载体。结果显示,BcGME基因包含一个长度为1137 bp的开放式阅读框(ORF),编码379个氨基酸。推测其蛋白理论相对分子质量为42.97×103,理论等电点pI值为5.84,分子式为C1907H2949N519O570S22,属于不稳定蛋白。总平均疏水指数为-0.440,属于亲水蛋白,亚细胞定位预测该蛋白分布在细胞质中。BcGME氨基酸序列与十字花科大部分植物的相似性为90%以上,不结球白菜BcGME与十字花科的大白菜、甘蓝型油菜、拟南芥相应蛋白的进化距离较近。并且不结球白菜和拟南芥GME蛋白三级结构相似,推测BcGME基因在抗坏血酸合成途径中起到关键作用。
[Abstract]:L-Ascorbic ascorbic acid, also known as vitamin C, is a kind of water-soluble antioxidant, Gallie 2013, which is widely found in animals and plants. The AsA synthesized in plants can not only make the human and some animals that can't synthesize AsA obtain rich vitamin C sources, but also maintain the normal growth and metabolism of plants (Anhua 2004. In recent years, increasing the AsA content of crops by genetic engineering technology is not only helpful to obtain higher levels of vitamin C in human body, but also has an important effect on enhancing the resistance of plants and improving the nutritional quality of agricultural products. In this paper, the relationship between ascorbic acid content and gene expression in non-heading cabbage under stress and hormone treatment was investigated, which laid a theoretical foundation for quality breeding of non-heading Chinese cabbage. The main results of this paper are as follows: 1: 1. The purpose of this study was to find out the changes of ascorbic acid content in non-heading Chinese cabbage under stress and to find out the key genes to regulate the accumulation of ascorbic acid content. The content of AsA and the expression of genes related to metabolism were determined by hydrogen peroxide stress and water flooding stress. The results showed that the AsA content increased first and then decreased under both treatments, and the change trend of GME and GPP gene expression under hydrogen peroxide stress was consistent with that of AsA content. However, the change trend of GME and VTC2 gene expression was the same as that of AsA content under waterlogging stress, indicating that GME gene may be the key gene. 2. In order to understand the changes of ascorbic acid content in Chinese cabbage without hormone treatment and to find out the key genes to regulate the change of AsA content, 'Suzhou Qing' was treated with abscisic acid (Abscisic acid) and ethylene (200 渭 mol L ~ (-1), respectively, in order to understand the changes of ascorbic acid content in Chinese cabbage without hormone treatment. The results showed that the change trend of AsA content was different after different concentration of ABA-treated. The results of gene expression analysis showed that the change trend of AsA gene expression was the same as that of AsA content in the low concentration ABA 10 渭 mol L ~ (-1) 50 渭 mol ~ (-1) and 100 渭 mol L ~ (-1) groups. After ethylene treatment, the AsA content increased first and then decreased. The change trend of GPP gene expression in ethylene treated with 10 渭 mol L-1 and 50 渭 mol L-1 was consistent with that of AsA content. However, the change trend of PMM and GPP gene expression in ethylene treated with 100 渭 mol L-1 and 200 渭 mol L-1 was the same as that of AsA content. Therefore, it is suggested that PMM and GPP genes may be the key genes of AsA biosynthesis pathway under abscisic acid and ethylene treatment, respectively. The full length of BcGE gene was cloned homologous from 'Suzhou Qing', and its amino acid sequence was analyzed by bioinformatics, and BcGME-PCVA silencing vector was constructed. The results showed that the BcGME gene contained an open reading frame of 1137 BP encoding 379 amino acids. The theoretical molecular weight of the protein is 42.97 脳 10 ~ 3, the theoretical isoelectric point Pi is 5.84, and the molecular formula is C1907H2949N519O570S22, which is an unstable protein. The total average hydrophobic index was -0.440, which belonged to hydrophilic protein. The similarity between the amino acid sequence of BcGME and most plants of Cruciferae was more than 90%, and the similarity between BcGME and Brassica campestris was more than 90%, the subcellular localization predicted that the amino acid sequence of BcGME was more than 90% similar to that of Brassica chinensis. In Brassica napus, the corresponding proteins of Arabidopsis thaliana were close to each other. The tertiary structure of GME protein in non-heading Chinese cabbage and Arabidopsis thaliana is similar. It is suggested that BcGME gene plays a key role in ascorbic acid synthesis pathway.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S634.3

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