【作者】 Wirat Rerksiri；

【导师】 陈信波；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 随着全球气候变暖,高温胁迫(热害)已成为全球范围的一个严重影响植物的生长和农作物产量的主要非生物逆境因子。微阵列技术已广泛应用于系统研究植物对复杂的不利环境因子变化的响应机制。微阵列技术可高效获取组织特异性和逆境响应特异表达基因。本研究微阵列分析技术对不同时间高温胁迫下水稻减数分裂后期幼穗进行了全基因组水平表达谱分析以获取高温胁迫响应基因信息。高效逆境特异响应启动子可用于驱动耐逆相关基因的高效表达以提高植物耐逆性。本研究从已有水稻微阵列数据筛选出来6个非生物胁迫应答基因,进一步利用定量PCR分析了这6个基因的在不同的器官和非生物逆境胁迫下表达模式；对其中3个胁迫诱导基因进行了启动子元件分析和启动子驱动的GUS表达分析。主要研究结果如下：1.利用Agilent44K水稻芯片获得了不同时间高温胁迫下水稻减数分裂后期幼穗的全基因组水平表达谱。获得了1815个水稻幼穗高温胁迫下差异表达基因进行了分析和评价。2.GO分类和基因表达模式分析结果表明,水稻穗中的热响应(HR)调控基因主要涉及转录因子、蛋白质修饰和降解、植物激素响应因子、受体激酶,钙相关蛋白质等。热激响应调控相关的转录因子家族主要包括bZIP、WRKY、NAC、C2H2、 MYB、MADS、bHLH和AP2/ERF家族。在高温胁迫下这些家族的基因大部分受到了不同程度的上调。3.基因表达谱分析表明：在高温胁迫下水稻幼穗中活性氧(ROS)系统相关基因的复杂的变化有助于保持ROS的平衡。高温胁迫促进了泛素蛋白酶体系统(UPS)基因表达。一些泛素-蛋白连接酶E3家族基因在高温胁迫下得到不同程度的上调,表明在幼穗高温响应和胁迫应对中发挥重要作用。4.水稻穗热响应的代谢相关的基因主要涉及细胞壁的形成和脂质代谢,特别是次生代谢途径。大多数代谢相关基因在高温胁迫下被一定程度下调,这种代谢抑制的将有助于植物应对逆境条件。5.从逆境胁迫下水稻微阵列分析数据中筛选除了6个显著高温诱导表达基因。采用定量PCR方法对这6个基因在不同组织器官和高温、盐胁迫聚乙二醇(PEG)和低温胁迫以及和脱落酸(ABA)处理下的表达特征进行了分析。6个基因的表达都受高温显著诱导,对盐胁迫PEG和ABA有一定程度响应,但对低温胁迫几乎不响应。在正常生长条件下6个基因在不同组织器官均有不同程度表达。6.利用PlantCARE and PLACE在线软件对3个高温诱导基因的启动子(OsHsfB2c,PM19p和HSP90p)进行了启动子元件分析,发现3个启动子都存在一些逆境响应相关元件,如G-box, GC-motif, HSE and ABRE。利用这3个启动子驱动GUS报告基因在转基因水稻中表达,进行了高温和干旱胁迫下转基因水稻幼穗和叶片中的GUS基因表达、组织化学染色和GUS酶活性分析。在高温胁迫下3个启动子的转基因水稻叶片和幼穗中都表现出很高的GUS活性,以OsHsfB2c和MP19p的幼穗在高温下的活性更高。实验结果表明OsHsfB2c和PM19启动子具有显著高温诱导特性,对其启动子的顺式元件的进一步鉴定和改造有可能获得在植物基因工程应用中具有重要价值的高温诱导型启动子。更多还原

【Abstract】 With the increasing global warming, heat stress is becoming a major abiotic stress limiting plant growth and productivity around the world. Microarray has been widely used to systematically investigate the molecular reactions by which plants respond and adapt to complicated environment. Microarray was also applied to profile the tissue-specific and stress-specific gene expression. In present study, a time course gene expression profile of post-meiosis rice panicle under heat stress was performed by microarray to uncover the genome-wide gene response to heat stress. Stress-inducible promoters are considered to be ideal for driving specific gene expression for increased abiotic stress tolerance. Six highly heat responsive genes were identified from microarray data for their promoter analysis. The expression patterns of these six genes in different rice organs and under abiotic stresses were further confirmed by quantitative RT-PCR and three highly stress-inducible genes were selected for promoter-driven GUS expression analysis under heat and drought treatments in panicles and flag leaves. The major results are as followed:1. A time course whole genome rice gene expression profile in post-meiosis rice panicle under heat stress was obtained using Agilent44K rice microarray. A total of1815differentially expressed genes (DEGs) in rice panicle under heat treatment were identified.2. The results from GO retrieve and gene expression pattern analysis showed that the heat-responsive (HR) regulation-related genes in rice panicle are mainly classified as transcription factors, protein modification and degradation, phytohormones responsive factors, receptor kinases, and calcium-related proteins. The HR regulation-related transcription factors consist of bZIP, WRKY, NAC, C2H2, MYB, MADS, bHLH, and AP2/ERF families and more than half of these genes were up-regulated to a different extent under high temperature.3. The expression profile analysis showed that the complicated changes of ROS-related genes in rice panicle under heat stress are important in maintaining ROS balance. Ubiquitin-proteasome system (UPS) was enhanced under heat stress. HR UPS-related genes from E3families were up-regulated to different extent upon heat shock in rice panicle, suggesting that the UPS-related genes play important positive roles in panicle response to heat.4. Metabolism-related HR genes in rice panicle were mainly involved in cell wall formation, lipid metabolism and especially in secondary metabolisms. Most metabolism- related genes were slightly repressed under heat treatment, which suggested that the inhibition of metabolism in rice panicle under high temperature would be helpful for plant response to adverse environments.5. Six highly heat responsive genes were identified from microarray data for their promoter analysis. The expression pattern of these six genes in different rice organs and under abiotic stresses were analyzed by quantitative RT-PCR. Expression of these six genes were highly heat inducible, moderately responded to salt stress, polyethylene glycol (PEG) and abscisic acid (ABA), but little affected to cold treatment. Under normal conditions, expression of the six genes was detected in all the organs analyzed but at different levels.6. The promoter analysis of the three highly heat-inducible genes (OsHsfB2cp, PM19p and HSP90p) by PlantCARE and PLACE showed some stress response-related elements, such as G-box, GC-motif, HSE and ABRE in the three promoters. These three promoters were used to drive GUS gene expression in rice. GUS gene expression, histochemical staining and GUS activities in panicles and flag leaves of the transgenic rice plants was analyzed under heat and drought treatments. The three promoters exhibited similar high activity lever in rice leaf under heat, but OsHsfB2cp and PM19p showed much higher activities in panicles under heat stress. Our work confirmed that the OsHsfB2c and PM19promoters were highly heat inducible and further characterization and reconstruction of cis-elements in their promoters could lead to the development of highly effective heat-inducible promoters for plant genetic engineering.更多还原