【作者】 孙延国；

【导师】 侯丙凯；

【作者基本信息】 山东大学 ， 遗传学， 2013， 硕士

【摘要】 植物对非生物胁迫的响应涉及一系列复杂的生理生化过程,往往是多基因相互作用的结果,目前已有的发现可能只是反映整个耐逆机制中的某些方面,因此,从新的角度研究植物耐逆性,可以对耐逆机制有一个多方位的认识,更好的理解植物的耐逆机制。当前对植物耐逆的研究主要集中在耐盐、耐旱等方面。当植物遭遇逆境时,为了使植物在胁迫条件下具有更大的灵活性,植物体内经常发生小分子的修饰作用,这其中,由糖基转移酶催化的糖基化修饰在调控各种小分子的溶解度、稳定性及生物活性中扮演了非常重要的角色。因此,植物糖基转移酶与植物耐逆性息息相关。糖基转移酶是专门负责催化糖基化修饰的酶类,它将活性糖基从供体转移到受体分子上,从而改变植物分子的生物活性、水溶性、在细胞内和整体植株的转运特性、亚细胞定位以及与受体的相互识别与结合特性等,因此,糖基转移酶在维持植物细胞代谢平衡、调节植物正常生长发育等方面有重要意义。近年来针对植物糖基转移酶尤其是拟南芥家族1糖基转移酶开展的许多研究,证明了植物糖基转移酶的诸多生物学功能。最近有研究证明植物糖基转移酶还参与调控植物应对非生物胁迫的反应过程,但其具体作用机理还不清楚,糖基转移酶参与植物耐逆过程的分子机制还需要人们去深入探索。在本实验室前期的研究中,我们发现拟南芥糖基转移酶基因UGT85A5受NaCl诱导表达,暗示了该基因可能参与植物耐盐反应,因此对其功能进行了深入的研究。另外,我们通过芯片数据,筛选了三个基因UGT85A2.UGT76E1. UGT73B1作为候选耐逆基因进行研究。本论文运用分子生物学和反向遗传学等研究手段,对以上基因的功能进行了分析,试图通过拟南芥和烟草等模式植物,探讨植物糖基转移酶与耐盐、耐旱作用之间的关系。论文主要内容与结果如下：1.首次证明了糖基转移酶基因UGT85A5具有增强植物耐盐性的功能作用。对UGT85A5转基因烟草进行了一系列耐盐实验分析,发现在盐胁迫条件下,转基因烟草比野生型烟草有更高的种子萌发率、更好的生长状态及更少的叶绿素损失,初步证明了UGT85A5增强植物耐盐性的作用。对烟草中的一系列耐盐生理指标进行了测定。结果表明：在盐胁迫处理下,UGT85A5转基因烟草比野生型烟草积累了更多的脯氨酸和可溶性糖,而积累了更少的丙二醛,具有更低的Na-/K+比率。这些结果农明了UGT85A5转基因烟草在盐胁迫条件下能更好地保护细胞结构,受到更少的伤害,表现出更高的耐盐性。对烟草中一些耐盐相关基因的表达水平进行了分析。结果表明：在盐胁迫处理下,UGT85A5转基因烟草中一些碳水化合物代谢相关基因SS、SPS、HT和编码一种保护蛋白的基因ERD10C的表达量都比野生型烟草中的明显高,这与之前测定的生理指标数据相符合。然后又对UGT85A5基因的启动子区序列进行了分析,发现在启动子区有许多已被报道与逆境响应相关的转录因子结合位点,如G-box序列、ACGT元件、GATA序列和ABRE结合位点等,表明了UGT85A5基因可能是植物调控逆境响应网络的一个目标基因。这些结果表明UGT85A5基因通过与上游及下游基因的相互作用调控植物的耐盐反应。2.获得了三个糖基转移酶基因的拟南芥过表达株系与突变株系,并对其进行了初步的耐逆实验分析。利用RT-PCR方法从野生型拟南芥(Col-0)克隆到三个基因UGT85A2、 UGT76E1、UGT73B1的cDNA全长序列,分别构建植物表达载体,通过农杆菌浸染、筛选、鉴定等过程获得了这三个基因的拟南芥过表达株系；通过三引物PCR方法获得了拟南芥UGT73B1基因的T-DNA插入纯合突变体。对以上材料进行了初步的耐盐、耐旱实验分析。综上,本论文通过一系列实验首次证明了糖基转移酶基因UGT85A5具有增强植物耐盐性的功能作用,丰富了植物的耐逆基因资源和耐逆机制。同时,还对另外三个糖基转移酶基因进行了初步的耐逆实验分析。然而,对于植物糖基转移酶基因参与植物耐逆过程的详细的分子机理还需要深入探索。更多还原

【Abstract】 The responses of plants to abiotic stress are involved in a complex series of physiological and biochemical processes, and they are often the results of interactions among multiple genes. Until now what we have found may simply reflect some aspects of the whole abiotic stress-tolerant mechanisms of plants. Therefore, studying the plant tolerance to abiotic stress from a new angle could help us to better understand plant abiotic stress tolerance mechanisms in a comprehensive level. Current researches on plant tolerance to abiotic stress mainly focus on salt and drought stress tolerance. To endow the plants with greater flexibility and plasticity under stress conditions, it is essential to make dramatic modifications to a variety of small molecules. In particular, glycosylation catalyzed by a superfamily of glycosyltransferases plays significant roles in modulating the solubility, stability, bioavailability and bioactivity of various small molecules. Thus, plant glycosyltransferases are closely related to plant abiotic stress tolerance.Glycosyltransferases are enzymes responsible for glycosylation of plant compounds, which catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, thus change their biological activity, water-solubility, transport characteristics, subcellular localization and binding properties with receptors. Therefore, glycosyltransferases play significant roles in maintaining cell homeostasis, regulating plant growth and development. Researches on plant glycosyltransferases, especially the family1glycosyltransferases of Arabidopsis thaliana in recent years, have demonstrated the wide range of biological functions of plant glycosyltransferases. Recent studies have shown that plant glycosyltransferases were involved in the regulation of plant responses to abiotic stresses, but their specific mechanisms of action remain to be determined. In our previous study, it was found that Arabidopsis glycosyltransferase gene UGT85A5was significantly induced by salt stress, indicating that it was involved in plant salt tolerance. In this thesis, we use molecular biology and reverse genetics methods to study the function of UGT85A5gene, trying to investigate the relationship between plant glycosyltransferase and stress tolerance through the model plants Arabidopsis thaliana and tobacco. In addition, we selected additional three glycosyltransferase genes (UGT85A2, UGT76E1, UGT73B1) as candidate stress-tolerant genes through microarray data, and studied their functions in stress tolerance. The main contents and results of this study are summarized as follows:1. The glycosyltransferase gene UGT85A5was for the first time demonstrated to play a significant role in enhancing plant salt tolerance.The results of a series of salt stress experiments showed that there were higher seed germination rates, better plant growth and less chlorophyll loss in UGT85A5transgenic tobacco compared to wild type plants under salt stress, suggesting the role of UGT85A5in enhancing plant salt tolerance.A series of salt-related physiological index were determined in transgenic tobacco. The results showed that the accumulations of proline and soluble sugars were increased, but the malondialdehyde accumulation and Na+/K+ratio were decreases in UGT85A5-expressing transgenic tobacco compared to wild type plants, indicating that UGT85A5transgenic tobacco could better protect the cell structure from damage under salt stress condition.The expression level of several salt-related genes was analyzed in tobacco. During salt stress, expression of some carbohydrate metabolism-related genes including those for sucrose synthase, sucrose-phosphate synthase, hexose transporter and a group2LEA protein were obviously upregulated in UG785A5-expressing transgenic plants compared with wild type controls, which were correlated with the physiological index described above. Furthermore, we analyzed the UGT85A5promoter and found that it has many binding sites of transcription factors responding to environment signals, such as ABRE binding site motif (ABF binding site), G-box promoter motif and the ACGT elements, which had been reported to be involved in responses to dehydration and ABA treatment. All these results suggest that UGT85A5may be a target of regulatory networks that control abiotic stress responses and paly a role through its interactions with upstream and downstream genes.2. The Arabidopsis overexpression lines and mutant lines of additional three candidate stress-tolerant glycosyltransferases genes were obtained, and stress tolerance analyses toward these materials were carried out.The full-length cDNA of UGT85A2, UGT76E1and UGT73B1were cloned from Arabidopsis thaliana using RT-PCR method, and their plant expression vectors were constructed. The Arabidopsis overexpression plants of these three genes were obtained through the Agrobacteium-mediaXed genetic transformation, kanamycin resistance screening and RT-PCR detection. The T-DNA insertion mutation homozygote of UGT73B1was identified using three-prime PCR method. Salt and drought stress tolerance analyses using these materials were carried out.In summary, this research demonstrated for the first time that the Arabidopsis glycosyltransferase gene UGT85A5plays a significant role in enhancing plant salt tolerance, thus providing a new gene for the crop breeding. In addition, brief stress tolerance analyses of the other three glycosyltransferase genes were also carried out. However, the detailed molecular mechanisms involved in plant abiotic stress tolerance of glycosyltransferases genes need further investigation.更多还原