【作者】 张莹；

【导师】 田振东；

【作者基本信息】 华中农业大学 ， 蔬菜学， 2009， 硕士

【摘要】 乙烯应答因子(Ethylene Responsive Factors,ERFs)是植物特有的转录因子,在植物生长发育和环境胁迫应答中起重要作用。ERF转录因子通过与病程相关基因(PR)启动子中GCC-box的结合来调控PR基因来提高植物对病害的抗性。ERF转录因子还可结合非GCC-box顺式作用元件参与植物对低温、干旱和ABA等非生物胁迫相关的应答过程。StERF3是本实验室克隆的与晚疫病抗性相关的抑制型(含有EAR-motif)转录调控基因。能够被SA、MJ和ETH诱导表达,该基因可能作为SA、JA和ET信号传导途径节点调控基因来调控PR基因表达,从而引起马铃薯对晚疫病菌的防卫反应。本研究进一步利用基因超量表达和干涉技术、启动子顺式作用元件分析以及下游调控基因分析,较系统地研究了StERF3在马铃薯抗晚疫病和非生物胁迫中的作用。主要结果如下:1.构建了StERF3超量表达载体,转化了E3(垂直抗性)和Jx(感病)两个马铃薯品种,分别获得E3超量表达株系34个,Jx超量表达株系18个。构建了特异干涉和非特异干涉表达载体,转化并获得E3特异干涉株系26个、非特异干涉株系21个,Jx特异干涉株系5个、非特异干涉株系4个。Southern杂交分析显示大部分株系呈阳性,拷贝数1-4。2.转基因植株RT-PCR基因表达分析显示,超量表达株系中StERF3的表达量高于E3和Jx对照;特异干涉和非特异干涉转基因株系中的表达量低于对照中的表达量。特异干涉转基因株系中,下游PR1、PR1b、PR4b、CHIB基因的表达量增强。在非特异干涉转基因植株中,StERF3被沉默的同时,与之同源的StEREBP1、CIP353和STWAAEIRD AP2/EREBP ERF类家族基因也被沉默。3.超量表达和干涉E3转化株系晚疫病原菌接种结果表明,9个超量表达株系的病斑扩展速率明显大于对照病斑扩展速率,占E3超量表达株系总接种株系数的36%;12个特异干涉株系的病斑扩展速率明显小于对照的病斑扩展速率,占E3特异干涉接种株系数的46.2%。4.150 mM NaCl盐胁迫条件下,Jx超量表达和特异干涉株系生长均受到明显抑制,但超量表达株系比干涉株系更为严重;4℃低温处理的E3超量表达株系顶端出现坏死、萎蔫,而特异干涉株系生长正常。表明干涉株系抗逆性有一定程度的提高。5.通过hiTAIL-PCR方法,在3个马铃薯品种中克隆了4条StERF3启动子序列,并对长度为1214 bp的启动子进行详细分析。除存在多个启动子结构元件之外,该启动子中存在多种与生物和非生物胁迫有关的顺式作用元件,如:应答ABA和干旱胁迫的pyrimidine-box,应答低温胁迫的LTRE-1,应答病程和盐诱导的GT-1及ABRE、MYBCORE、MYC、W-box、GCC-box等。综上所述,特异干涉StERF3转基因株系的晚疫病抗性增强,同时对盐胁迫和低温的耐受性有一定程度的提高。StERF3作为转录抑制子,可能通过负调控抗病和逆境相关基因的表达,从而调节马铃薯对生物和非生物胁迫的应答。更多还原

【Abstract】 The ERFs (Ethylene-Responsive Factors), are unique transcript factor of plants, play an important role in plant growth and stress response. It can improve plant resistance to diseases through interaction with GCC box which present in promoters of many pathogenesis-related (PR) genes. In addition, ERFs can response to abiotic stress, such as cold, drought, ABA signaling pathway through interaction with non GCC box containing genes.StERF3 is an EAR-motif containing suppress transcription regulation gene associated with late blight resistance which was cloned by our lab previously. It could be induced by SA, JA and ETH, and was conferred that it may act as an important element among the network of signal pathways to modulate PR genes expression which lead to the defense response to Phytophthora infestans. In this study we further investigated its functions against late blight resistance and abiotic stress through the overexpression and RNA interference methods, promoter cis elements and downstream regulated genes analysis. The main findings are as follows:1. Overexpression vector of StERF3 was constructed and transformed into two potato varieties E3 and Jx. Thirty four E3 and 18 Jx overexpression transgenic lines was obtained separately. At the same time, RNA specific interference and RNA family interference vectors was also constructed. We got 26 RNA specific interference transgenic lines and 21 family interference transgenic lines of E3, 5 specific interference and 4 family interference transgenic lines of Jx. Southern blot analysis indicated that most transgenic lines are positive and gene copy number ranged frome 1 to 4.2. RT-PCR analysis of transgenic plant showed that the expression of StERF3 in over-expression transgenic lines are higher than that in E3 and Jx control, but with lower level in RNA specific interference and RNA family interference transgenic lines compared with control. Expression of PR1、PR1b、PR4b、CHIB, the downstream gene of StERF3 with GCC-box containing in their promoters, were increased in RNA specific interference transgenic lines. In RNA family interference transgenic lines, besides StERF3 interference, the AP2/EREBP family homologous gene StEREBP1, CIP353 and STWAAEIRD shown silenced in different degree.3. Detached leaves inoculation of P. infestans showed that the lesion growth rate of 9 overexpression transgenic lines are significantly higher than that of control which hold 36% of the total inoculated overexpression transgenic lines. The lesion growth rate of 12 RNA specific interference transgenic lines are lower than that of control which stand 46.2% of the total inoculated RNA specific interference transgenic lines.4. Under 150 mM NaCl salt stress condition, the growth of overexpression and RNA specific interference transgenic lines were both inhibited, but the former is more serious than that of latter. After 4℃cold treatment, the top of E3 overexpression transgenic lines are necrosis and wilting, while specific interference transgenic lines grows well as normal. This indicated that resistance of specific interference transgenic lines increased in a certain degree.5. Using hiTAIL-PCR, 4 StERF3 promoter sequences were cloned, and one of 1214 bp in length sequence was analyzed in detail. In addition to a number of basal promoter structural elements, there are a wide range of biotic and abiotic stress-related cis elements in it, such as pyrimidine-box response to ABA and drought stress, LTRE-1 response to cold, GT-1,ABRE、MYBCORE、MYC、W-box、GCC-box response to disease and salt stress.To sum up, the late blight resistance level of StERF3 specific interference transgenic lines are increased; at the same time salt stress and low-temperature tolerance are raised at a certain extent. Thus, it can be inferred that StERF3, as a transcription inhibitor, regulated biotic resistant and abiotic stress response in potato mainly by negativly regulate disease-resistant and stress-related genes.更多还原