KMS XINJIANG INSTITUTE OF ECOLOGY AND GEOGRAPHY，CAS
| 胡杨BR信号通路中转录因子PeBZRI/PeBESI基因功能研究 | |
| 李涛涛 | |
| Subtype | 硕士 |
| Thesis Advisor | 姚银安 |
| 2016 | |
| Degree Grantor | 中国科学院大学 |
| Place of Conferral | 北京 |
| Degree Discipline | 植物学 |
| Keyword | 胡杨 非生物胁迫 Pebzr1/pebes1基因 功能验证 |
| Abstract | BR（Brassinosteroid）作为植物生长的必需激素，在调控植物的生长、发育和保护植物免受环境胁迫方面具有非常重要的作用，如促进细胞伸长和分裂，提高花粉育性，调节细胞壁结构和增强抗逆性等。目前BR 信号转导的研究主要集中于拟南芥、水稻、番茄等一年生草本植物，对于广泛存在于自然界的木本植物，其BR信号转导过程中的关键基因功能鲜有研究。本研究主要对胡杨(P. euphratica) BR信号通路中转录因子PeBZR1/PeBES1基因的功能进行了初步探究，以期初步阐明PeBZR1/PeBES1基因在抗逆能力方面的作用，同时也为利用PeBZR1/PeBES1基因进行改良林木和农作物提供一定的理论基础，研究结果如下： （1）通过对干旱、盐和铜胁迫下胡杨和俄罗斯杨BZR1/BES1基因表达模式的分析发现，三种胁迫都能影响BZR1/BES1基因的表达，两个树种之间差异较大；在盐胁迫下，胡杨的BZR1/BES1的表达量保持相对稳定，俄罗斯杨表达量逐渐升高后降低。通过对组织表达差异分析发现BZR1/BES1基因在根茎叶中均有表达，BZR1基因在胡杨的根茎叶中表达量差异小，俄罗斯杨根中最少，叶片其次，茎中最多，BES1基因在胡杨中变化较小，俄罗斯杨的叶片中表达量显著高于其他组织。 （2）通过生物信息学预测PeBZR1/PeBES1基因编码蛋白的各种理化性质，构建了BZR1/BES1基因的系统进化树，并通过烟草原生质体观察两个转录因子的亚细胞定位，发现两个都在细胞核内表达；分析了PeBZR1/PeBES1启动子的功能元件，而且从胡杨中克隆出PeBZR1/PeBES1基因，并通过overlapping PCR得到了两个基因的功能获得型Pebzr1-1D和Pebes1-1D载体。 （3）将过表达载体35S::PeBZR1和35S::PeBES1转入烟草，通过对不同浓度盐胁迫下的生理生化指标分析发现：在盐胁迫下，与对照相比过表达的转基因烟草株系都显著提高了烟草的SOD、POD和CAT酶的活性，不同盐浓度下程度不同，各转基因株系之间也存在差异；渗透调节物质脯氨酸和可溶性糖的含量也变化显著，PeBZR1转基因株系明显高于PeBES1；与对照相比转PeBZR1/PeBES1基因株系的过氧化氢含量降低，可溶性蛋白含量升高；MDA含量也有降低的趋势；而NtNHX1和NtHAK1基因的表达量在不同浓度盐胁迫下差异显著，转基因株系的表达量显著高于对照，说明PeBZR1/PeBES1转录因子上调了这两个耐盐基因的表达，以上说明了转PeBZR1/PeBES1基因的烟草耐盐性明显高于对照，促进了烟草在盐胁迫下的正常生长, 转基因种子的在不同胁迫下的萌发实验也验证了转基因种子能够提高胁迫条件下的萌发率、根长及α—淀粉酶活力。 |
| Other Abstract | Brassinosteroids (BR) are essential hormone for plant growth and development, and also protect plants from a variety of environment strsses, such as cell elongation and division, pollen fertility, photomorphogenesis, adjusting the structure of cell wall and enhancing resistance. Current research focused on annual herbs, such as Arabidopsis thaliana, rice, Solanum lycopersicum and so on, For widely distributed woody plants in nature is less known, the study on Populus (P. euphratica) BR signaling pathway transcription factor PeBZR1 / PeBES1 is a preliminary inquiry to elucidated the role of PeBZR1 / PeBES1 gene in Populus BR signal transduction and improving capabilities of plant defense, providing a theoretical basis knowledge of BR signal in other woody plants, and making us have more accurate understanding the difference between woody and herbaceous inbiosynthesis and metabolic mechanisms of the BR signal. It also provides a theoretical basis for the using of PeBZR1 / PeBES1 gene in modified trees and crops. The results are as follows: (1) P. euphratica and P. russkii BZR1 / BES1 gene expression have larger difference under drought, salt and copper stress. The expression of P. euphratica BZR1 / BES1 gene relatively stable and P. russkii has gradually increased under salt stress. Through analysis gene expression of differential tissue, BZR1/BES1 gene is active in stems, leaves and roots of the two Populus. BZR1 gene expression has little difference in different tissues of P. euphratica, the BZR1 gene expression of P.russkiiis roots least, leaves medium, stems up. BES1 gene expression of P. euphraticaia is smaller changer, BES1 gene expression of leaves of P. russkiiis significantly higher than other organizations. (2) By bioinformatic prediction the physical and chemical properties of PeBZR1 / PeBES1 protein, and observed the subcellular localization of two transcription factors expression in Neuclear through tobacco protoplasts. Moreover, constructed phylogenetic tree of PeBZR1 / PeBES1 gene and analysis functional element of the promoter, and obtained two gain of the functions vector PeBZR1-1D and PeBES1-1Dfrom PeBZR1 / PeBES1 by overlapping PCR. (3) The over-expression vector 35S::PeBZR1 and 35S::PeBES1 was transformed into tobacco leaves, analysisthe outcome of physiological and biochemical indexes under different concentrations of salt. Under different salt concentrations, the over-expression of transgenic tobacco lines have significantly increased in SOD, POD and CAT enzyme activity compared with the control, and the degree are differences in transgenic lines; osmotic adjustment including proline and soluble sugar content also showed a significant increase, PeBZR1 transgenic lines were significantly higher than PeBES1, MDA content decreased, hydrogen peroxide content decreased and soluble protein content increased of the PeBZR1 / PeBES1 transgenic lines compared with the control, and the expression of NtNHX1 and NtHAK1 genes have significantly different level between transgenic lines and the control in different concentrations of salt stress. it may indicat that PeBZR1 / PeBES1 transcription factors indirect regulating the expression of these two salt tolerance gene.The result revealstransforpmeBZR1 / PeBES1 gene line of tobacco has significantly higher than the control in the salt tolerance and promote the normal growth of tobacco under salt stress.Transgenic seeds germination experiments also proved genetically improved seed germination rate, root length and α-amylase under different stress conditions. |
| Subject Area | 植物学 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | http://ir.xjlas.org/handle/365004/14704 |
| Collection | 研究系统_荒漠环境研究室 |
| Affiliation | 中科院新疆生态与地理研究所 |
| Recommended Citation GB/T 7714 | 李涛涛. 胡杨BR信号通路中转录因子PeBZRI/PeBESI基因功能研究[D]. 北京. 中国科学院大学,2016. |
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