【作者】 贾芝琪；

【导师】 杜永臣；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2008， 博士

【摘要】 晚疫病是危害茄科作物马铃薯和番茄最严重的病害之一,对番茄和马铃薯生产造成巨大的经济损失。近年来,马铃薯抗晚疫病基因克隆取得了很快的进展,已经有来源于野生种Solanum. demissum的R1和R3a及来源于S. bulbocastanum的RB和Rpi-blb2、Rpi-blb3被克隆。由于晚疫病进化潜力高,所以新的抗病基因很快就被新的生理小种克服,限制了抗病基因的利用。相似的情况也出现在番茄抗晚疫病研究中,番茄有四个抗晚疫病基因被定位,但这四个基因的在田间的应用效果也十分有限。因此探索已克隆抗病基因的抗病机理,了解抗病信号传导途径就显得尤为重要,发现信号途径中的关键基因必将为晚疫病的防治开辟一个新的领域。抗病基因R3a和无毒基因Avr3a是晚疫病研究中第一对被克隆的符合基因对基因假说的R-Avr基因对,它们的克隆为我们研究抗晚疫病信号途径提供了基础。本论文主要的研究内容与结果如下:1.利用VIGS和瞬时表达技术研究已知的信号传导关键基因是否参与R3a-Avr3a的互作。通过构建已知抗病信号传导关键基因EDS1、NDR1、RAR1、SGT1、HSP90、NPR1、NtWRKY1、SIPK1的VIGS载体,以烟草作为模式系统沉默上述八个基因后,再利用瞬时表达技术在已沉默基因的烟草叶片中瞬时表达R3a和Avr3a,结果表明在SGT1和HSP90基因沉默的烟草植株中,R3a和Avr3a互作的HR被阻断,说明这两个基因参与了R3a与Avr3a的HR反应,在R3a的抗病信号途径中起重要作用。2.将抗晚疫病基因R3a转入番茄,获了对马铃薯晚疫病菌小种特异性抗性的转基因植株,说明R3a基因抗病信号传导途径在番茄和马铃薯中的保守性,也为利用番茄研究马铃薯的抗病基因R3a提供了证据。为了发现新的R3a与Avr3a互作过程中的关键基因,我们构建了一个用于研究晚疫病抗病信号传导的系统。在R3a转基因番茄后代中利用皮脂激素诱导系统（GVG系统）诱导表达与R3a相对应的无毒基因Avr3a,获得的MM-R3a-Avr3a转基因植株叶片在涂抹或喷施化学诱导剂地塞米松（DEX）的情况下能够诱导类似HR反应的产生,该系统能够用来研究构建突变体库,筛选HR反应被阻断的突变体,用于R3a与Avr3a互作信号途径的研究。3.快速筛选MM-R3a-Avr3a转基因植株的纯合株系是建立突变体库的重要前提。利用连接接头的PCR方法,扩增R3a基因在番茄基因组中插入位点的侧翼序列,该序列与NCBI数据库中位于第四号染色体的编号为SL_MboI-127L14的克隆有100%的相似性,说明R3a基因整合于番茄的第四号染色体上。Avr3a基因侧翼序列与SGN的番茄BAC末端数据库中编号为LE_HBa0167K15_SP6₁3180的序列具有100%的同源性,但该BAC末端序列定位于哪条染色体目前未知。利用R3a和Avr3a的侧翼序列和边界序列的扩增,在转基因的T1代筛选到两个基因都纯合的MM-R3a-Avr3a植株。4.为了研究抗晚疫病基因R3a能否应用于番茄,我们对22份番茄晚疫病菌中的Avr3a等位基因进行了分析,结果表明R3a基因可以应用于部分地区番茄的抗晚疫病基因工程育种;除此之外,我们还将转R3a基因番茄和转另外两个马铃薯抗晚疫病基因R1和Rpi-blb1/RB的番茄植株,接种番茄晚疫病菌的主流小种和强致病力小种,证明马铃薯的抗晚疫病基因在番茄中对番茄的晚疫病菌株均具有一定的抗病功能,尤其是马铃薯抗晚疫病基因RB能够对番茄致病力最强的菌株产生抗性,为番茄的抗晚疫病基因工程育种提供了新的途径。更多还原

【Abstract】 Late blight, caused by the oomycete Phytophthora infestans（Mont.）de Bary, is a devastating disease of two Solanaceous crops, tomato and potato. Over decades years efforts, five R genes have been cloned, such as R1 and R3a from Solanum demissum, RB （Rpi-blb1）, Rpi-blb2 and Rpi-blb3 from S. bulbocastanum. But as new virulent races emerged rapidly, these race-specific R genes provided only short-lived resistance in the field. The similar situations have occurred in tomato, four resistance genes have been mapped and deployed in cultivars but the effectiveness is limited because of the fast evolving P. infestans pathogen in the field. In-depth understanding of signal transduction mechanisms underlying potato R gene resistance may help to generate effective measures to control late blight. R3a from potato and Avr3a from P. infestans are the first pair of R-AVR genes which have been cloned in potato and P. infestans which is the basic theory of our research.The results were as follows:1. Constructed the VIGS vectors contain key signal transduction gene EDS1, NDR1, RAR1, SGT1, HSP90, NPR1, NtWRKY1, SIPK1 respectively. Transient expressed R3a and Avr3a gene in Nicotiana benthamiana after the VIGS gene silencing. Hypersensitive cell death was interrupted in SGT1 and HSP90 silenced plants which indicated that SGT1 and HSP90 participated in R3a and Avr3a interaction and played a key role in R3a resistance sinal pathway.2. Transgenic tomatoes contain R3a gene displayed race-specific resistance to P. infestans which verified the conservation of resistance signal pathway in tomato and potato, so it is feasible to use tomato as a model plant to research tomato late blight gene R3a. In order to find new genes that participate in R3a and Avr3a interaction, a novel transgenic tomato plant conferred potato late blight resistance gene R3a and glucocorticoid-inducible avirulence gene Avr3a was constructed in our study. MM-R3a-Avr3a plants showed specific cell death response induced by DEX. This system will be used to large-scale screen the mutants which HR were interrupted.3. In order to screen homozygous MM-R3a-Avr3a lines to construct the mutant library, we use modified adaptor ligation PCR method amplified R3a and Avr3a gene flanking sequences in tomato genome. The flanking sequence of R3a had 100% similarity with clone SL_MboI-127L14 on chromosomeⅣin NCBI database which indicated R3a gene integration in tomato genome on chromosomeⅣ. The flanking sequence of Avr3a had 100% similarity with a BAC end sequence LE_HBa0167K15_SP6₁3180 in SGN database, but until now we still didn’t know this BAC end sequence located on which chromosome. By flanking sequence and border sequence we screened out homozygous MM-R3a-Avr3a lines in T1 transgenic generation.4. We analyzed Avr3a alleles of 22 tomato late blight isolates which demostrated that R3a gene can be used in tomato reisitance breeding in some areas. Other two potato late blight genes R1 and Rpi-blb1/RB were also introduced into tomato. Transgenic plants were inoculated by tomato widly distribute isolates and the high virulence isolates and showed resistance to some tomato late bligt isolates, especially RB, resist the high virulence isolates of tomato which offered a new way for tomato late blight transgene breeding.更多还原