拟南芥Sep15-like基因耐逆功能及其作用机制

发布时间：2018-04-13 16:57

本文选题：Sep15-like + 抗逆　；参考：《山东大学》2017年硕士论文

【摘要】：作物在生长过程中受到非生物逆境的胁迫通常会导致细胞内氧化还原系统的失衡和内质网压力增大,从而影响了作物的生长和发育。因此鉴定抗逆相关基因及解析其作用机制并应用于育种工作,是提高作物产量的一个重要途径。在动物中,Sep15参与多种氧化还原反应、蛋白质折叠的质量控制等,但在植物中其同源基因的功能未见报道。通过测序公共数据库搜索得到了拟南芥、短柄草和小麦的Sep15-like的cDNA和gDNA序列,设计了特异性引物,克隆得到了全长Sep15-like基因序列。序列分析发现Sep15-like在不同植物中比较保守且具有高度相似的两个结构域:富含半胱氨酸的结构域和硫氧还蛋白样折叠结构域;亚细胞定位实验显示植物Sep15-like蛋白定位于内质网中;组织表达模式分析显示,植物Sep15-like在根和叶中的表达量相对较高;盐、旱胁迫下的诱导表达模式分析表明,植物Sep15-like在盐、旱胁迫下其表达水平明显上调。将AtSep15-like、BdSep15-like和TaSep15-like转入拟南芥中,并获得过表达株系。在萌发阶段,正常条件下,除突变体萌发略差外,野生型和过表达系的萌发情况基本一致,胁迫处理条件下,突变体萌发情况最差,过表达系好于野生型;在幼苗阶段,正常条件下,突变体生长受到抑制,而野生型和过表达系的生长状况基本一致,胁迫处理条件下,突变体的根长明显短于野生型,而过表达系相比于野生型根长增加显著,地上部分差异也比较显著。同样地,BdSep15-like和TaSep15-like转拟南芥过表达系也展现了类似的表型。通过对内质网压力相关Marker基因的测定和蛋白质羰基、巯基含量的测定,初步证明AtSep15-like基因在胁迫条件下可以有效缓解内质网压力。通过对ROS含量和ROS信号途径相关基因的分析,表明AtSep15-like对于ROS平衡具有显著的调控作用。遗传互补实验表明AtSep15-like通过AtUGGT发挥了抗逆作用。本论文的研究证明了植物Sep15-like在抗逆方面的生理功能及其与内质网压力和活性氧之间的关系,并可能通过UGGT发挥作用。
[Abstract]:The stress of abiotic stress during the growth of crops usually leads to the imbalance of intracellular redox system and the increase of endoplasmic reticulum pressure, which affects the growth and development of crops.Therefore, it is an important way to improve crop yield to identify the genes related to stress resistance, to analyze the mechanism of their action and to apply them to breeding.Sep15 is involved in many redox reactions and quality control of protein folding in animals, but the function of its homologous genes has not been reported in plants.The cDNA and gDNA sequences of Sep15-like of Arabidopsis thaliana, Stipa sativus and wheat were obtained by sequencing the common database. Specific primers were designed and the full-length Sep15-like gene sequence was cloned.Sequence analysis showed that Sep15-like was conserved and highly similar in different plants: cysteine-rich domain and thioredoxin like folding domain, subcellular localization experiments showed that plant Sep15-like protein was located in endoplasmic reticulum (ER).Tissue expression pattern analysis showed that the expression of plant Sep15-like was relatively high in roots and leaves, and the induced expression pattern under salt and drought stress showed that the expression level of plant Sep15-like was significantly up-regulated under salt and drought stress.AtSep15-like Bd Sep15-like and TaSep15-like were transferred into Arabidopsis thaliana and the expressed lines were obtained.In the germination stage, the germination of wild type and overexpression line was basically the same except for the slightly worse germination of mutant, and under stress, the germination of mutant was the worst, the over-expression line was better than wild type, and in seedling stage, the germination of wild type and overexpression line were better than that of wild type.Under normal conditions, the growth of mutant was inhibited, but the growth status of wild type and overexpression line was basically the same. Under stress treatment, the root length of mutant was significantly shorter than that of wild type, and the root length of overexpression line increased significantly compared with that of wild type.There was also significant difference in the above-ground part.Similarly, Bd Sep15-like and TaSep15-like-transformed Arabidopsis overexpression lines showed similar phenotypes.Through the determination of endoplasmic reticulum pressure-related Marker gene and protein carbonyl and sulfhydryl content, it was preliminarily proved that AtSep15-like gene could relieve endoplasmic reticulum pressure effectively under stress.The analysis of ROS content and ROS signaling pathway showed that AtSep15-like had a significant role in regulating ROS balance.Genetic complementation experiment showed that AtSep15-like played a role of stress resistance through AtUGGT.In this paper, the physiological function of plant Sep15-like in stress resistance and its relationship with endoplasmic reticulum pressure and reactive oxygen species were proved, which may be played by UGGT.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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