【作者】 王小燕；

【导师】 何水林；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 植物在长期的进化过程中逐渐形成了诱导型防御反应机制,使以消耗物质和能量为代价的防御反应限制在特定的时间或空间,以实现物质和能量的有效利用。防御反应基因应答逆境胁迫主要由防御反应基因启动子中的顺式作用元件与其相应的反式作用因子(转录因子)的识别和结合所控制,因此,分离、分析应答特定逆境的顺式作用元件不仅有利于进一步分离其相应的转录因子阐明诱导型防御反应的信号传递通路,还有利于应用这些元件构建诱导型人工合成启动子用于植物抗逆基因工程遗传改良。本研究对前人从双子叶植物中分离、鉴定出来的DRE盒、W盒和S盒元件在水稻中应答不同逆境的作用进行了较为系统的研究,主要结果如下:1、采用常规的酶切连接技术与gateway克隆技术相结合,构建了一套将DRE盒、W盒和S盒等参试顺式作用元件插入到CaMV35S核心启动子(+8bp—-46bp)的上游,控制GUS报告基因表达的植物表达载体,通过测序对上述载体进行了验证;2、利用农杆菌介导的遗传转化方法分别将上述载体转入粳稻日本晴中,获得了上述载体相应的转基因T0代植株,对上述T0代植株在温室栽培并进行自交纯合和潮霉素浸种筛选,分别获得其足够的T1和T2代株系。3、利用上述转基因株系的种子、植株或其衍生的抗性愈伤组织,采用低温、高温、盐胁迫、稻纵卷叶螟取食和稻瘟病接种等方法处理结合GUS组织化学染色的分析方法,分析了DRE盒、W盒和S盒元件在转基因水稻中对参试的不同逆境胁迫的应答,并分析了不同元件对上述逆境胁迫应答与几种内源激素信号之间的可能关系:实验发现,W盒可介导水稻对真菌性病害稻瘟病的应答,参与了对稻纵卷叶螟取食的应答,还可能与JA和SA信号途径关系。DRE盒驱动的GUS基因表达对各种逆境的应答很弱甚至没有表达,说明在单子叶植物水稻中顺式作用元件与反式作用因子之间的识别和互作与其侧翼序列有关。S盒可明显介导水稻对稻瘟病和稻纵卷叶螟的应答,对各种非生物逆境的应答水平不同,而对外源激素有一定的表达。4、选择对病原菌或诱发因子处理高效应答的元件,选择合适的拷贝数以及适宜的组合,构建含多个顺式作用元件组合(2×DRE+2×GCC+2×JERE+2×W)的诱导型启动子的表达载体,并通过农杆菌介导的遗传转化方法获得转基因T0代植株,并对抗性植株进行PCR分子鉴定。本研究的初步研究结果表明,在单子叶水稻和双子叶植物之间,顺式作用元件与其反式作用因子之间的识别和互作激活植物防御反应存在着一定程度的保守性,W盒和S盒在稻瘟病和稻纵卷叶螟等生物性逆境胁迫具有较强的应答作用,同时也对低温、高盐等非生物逆境胁迫具有不同程度的应答作用,暗示在其上游存在着复杂的信号通路的crosstalk,可能与水杨酸、脱落酸等内源激素的参与有关。W盒和S盒等顺式作用元件可以不依赖于其侧翼序列在不同的遗传背景下单独发挥作用,表明这类顺式作用原件在诱导型合成启动子的构建中具有十分重要的应用价值。更多还原

【Abstract】 Abstract: Plants in the long process of evolution has gradually formed the inducible defense reaction mechanism,which makes the defense response for the cost of materials and energy consumption limited to a specific time or space in order to achieve effective utilization of material and energy.Defense response genes response to adversity stress is mainly controlled by the identification and integration of the cis-acting elements and their corresponding trans-acting factors (transcription factors) in the defense response gene.So, the separation and analysis of the cis-acting element of the response to specific adversity is not only conducive to further separation of the corresponding transcription factor inducible defense response to clarify the signaling transduction pathway, is also beneficial to use these components to build inducible synthetic promoter for the genetic improvement of plant resistance Reverse genetic engineering. This paper is a more systematic study,for the response to different stress role of the isolated and identified DRE , W and s-box components on predecessors from dicotyledonous plants in the rice. The main findings are as follows:1. In an ordinary endonuclease connection technology and gateway cloning techniques ,it builds a DRE, W and s-boxes, and other participants cis-acting elements inserted into the CaMV35S core promoter (+ 8bp—-46bp) upstream and controls the plant expression vector of GUS reporter gene expression, and tests the carrier by sequencing.2. Using Agrobacterium-mediated transformation methods to turn the carrier into japonica Nipponbare,it wins the vector corresponding transgenic T0 generation plants and makes T0 generation plants in the culture under-glass and makes them selfing、homozygous and hygromycin to be immersed for selecting,respectively requiring its inadequate T1 and T2 generation strains.3. Using the seeds and plants or its derivative resistance callus of these transgenic strains, at low temperature, high temperature and salinity, rice leaf roller feeding and rice blast vaccination and so on and together with the analysis methods of GUS histochemical stain,the author analyzes the response from the DRE, W and s-box components in transgenic rice on participants of different stress and analyzes the possible relationship between the response from different components to the stress and several internal endogeny hormone signals. The experiment found that W-box can be mediated the response from rice to fungus Magnaporthe grisea and, participated in a rice feeding response, and possibly with JA and SA signal pathway relationships. Gus expression driven by DRE box to the answer of the various abiotic stresses is different,,and it may be related to the upper reaches of the relationship between hormonal signals. S-box could significantly be mediated the response from rice blast and rice leaf roller ,and the level on a variety of abiotic stress response is different, while it is the external expression of a certain hormone.4. Selecting on pathogens bacteria or evoked inducer processing efficient answering components, and selecting the appropriate number of copies and appropriate combination to build the expression vector of inducible promoter containing more than one combination of CIS-acting elements (2×DRE + 2×GCC + 2×JERE + 2×W),and by Agrobacterium-mediated transformation method to obtain a transgenic plant T0 generation, identify confrontational plant by PCR.Preliminary findings indicate that between unifacial leaf rice and dicotyledonous plants, CIS-acting elements and trans-act factor between recognition and interaction activating plant defense responses exist a certain degree of conservative, W and s-box in the rice blast and rice leaf roller, and other biological adversity stress have strong response, at the same time they have different levels of response at low temperature, high salt and so abiotic stress,and this implied the existence of complex signaling pathways of crosstalk in its upper reaches ,and might have something to do with salicylic acid, Abscisic acid and other hormones involved. W and s-box, and other cis-acting elements can not rely on its flanking in different genetic backgrounds alone,and this indicates that this class of cis-acting original synthesis in inducible promoter of the construction is of great value.更多还原