【作者】 刘颖超；

【导师】 董金皋； 夏亦荠；

【作者基本信息】 河北农业大学 ， 植物病理学， 2004， 博士

【摘要】 本文对拟南芥不同生态型对不同病原菌的抗病性进行了初步研究，筛选出了对同一病原菌接种后抗病性反应表现不同的生态型，并对抗感表现不同的生态型接种后的抗性机制及抗性相关基因的表达进行了研究。 通过对8种病原菌的11个菌株及2种植物病原真菌毒素的反应，供试的12个生态型对甘蓝枯萎病菌(Fusarium campestris)、西瓜枯萎病菌(F.oxysporum f.sp.niveum)(3个菌株)、玉米大斑病菌(Setosphaeria turcica)99-2菌株、白粉病菌(Erysiphe cichoracearum)表现为非亲和反应；对白菜黑斑病菌(Alternaria brassicicola)、葡萄灰霉病菌(Botrytis cinerea)、番茄灰霉病菌(B.cinerea)、马铃薯晚疫病菌(Phytophthora infestans)、白菜霜霉病菌(Peronospora parasitica)，不同的生态型反应不同：其中，Col生态型对葡萄灰霉病菌(B.cinerea)、番茄灰霉病菌(B.cinerea)及白菜霜霉病菌(P.parasitica)表现为亲和反应；C24生态型对白菜霜霉病菌(P.parasitica)表现为亲和反应；NahG对葡萄灰霉病菌(B.cinerea)、番茄灰霉病菌(B.cinerea)表现为亲和反应；Est-1生态型、Bay-0生态型对白菜黑斑病菌(A.brassicicola)、葡萄灰霉病菌(B.cinerea)、番茄灰霉病菌(B.cinerea)、马铃薯晚疫病菌(P.infestans)和白菜霜霉病菌(P.parasitica)表现为亲和反应。用外源物质水杨酸和茉莉酸处理拟南芥与马铃薯晚疫病菌互作表现为亲和反应的生态型Est-1、Bay-0，提高了对马铃薯晚疫病菌的抗性，在接种部位只出现了侵染点或很小的坏死斑。 在相关防御酶系研究中发现，不同生态型的拟南芥在接种马铃薯晚疫病菌后其PAL、POD、PPO的活性和空白对照相比，均有不同程度的提高，而且抗病生态型Col酶活性提高的幅度及上升速度均高于感病生态型。在不同生态型的SOD比活性测定中，感病生态型接种7d内SOD活性迅速增加，到第9d迅速下降，抗病生态型Col在接种后SOD活性小幅度上升—下降—上升，接种第7d达到高峰。接种马铃薯晚疫病菌后，供试的3种生态型(Col、Est-1和Bay-0)中CAT酶活性都发生了变化，其中，Col在接种后酶活性基本是一直上升，在接种第7d时达到最大值，以后缓慢下降；而Est-1生态型和Bay-0生态型在接种后24h内酶活性变化不大，仅有小幅度下降，24h以后才开始逐渐上升，3～5d时达到最大值，以后下降，到第13d时，酶活性低于不接种对照，抗病生态型Col的CAT活性的最大值明显高于感病生态型Est-1和Bay-0。 在非酶类抗性相关物质研究中，抗病生态型和感病生态型接种马铃薯晚疫病菌后，各种抗性物质也有明显不同的变化。表现为：(1)接种马铃薯晚疫病菌后，感病生态型Est-1和Bay-0电导率变化比抗病生态型Col早，而且增加幅度也比抗病生态型Col大。(2)病原菌侵染可以引起细胞中游离脯氨酸含量的增加，抗病生态型增加幅度大于感病生态型。(3)丙二醛(MDA)作为膜脂过氧化的产物，在接种马铃薯晚疫病菌后供试的所有生态型都呈现上升趋势，抗病生态型Col接种后MDA值开始上升的时间比感病生态型早，但其最大值明显比感病生态型小。 利用优化的差异显示技术，对接种马铃薯晚疫病菌后不同拟南芥生态型差别表达的基因进行了分析，共获得了108条差别表达的基因片段，对差别表达基因片段的二次扩增产物进行了Northem杂交检测。结果表明，57个差异条带的二次扩增产物与对照或接种处理得到的cDNA杂交有阳性反应，阳性率为52.5%。对2个差异片段进行克隆并测序，获得了长度分别为269bp和466bp的2条cDNA序列。同源性分析表明，7一6序列(466bp)来自At3g47900.l基因，编码ACC合成酶;6一6序列(269bp)来自At5932405.1基因，其生物学作用、细胞组成及分子功能均未发表。 用反式Northem杂交检测抗病相关基因的表达。结果表明，特异于7个抗病相关基因的探针中，Col生态型的二次扩增产物与PR一3基因有杂交信号;Bay一0生态型与pR一l基因有杂交信号;Est一l生态型与At2932680，Atlg67980，poFI.2，PR一3不11PR一4基因有杂交信号。用DDRT-PCR与Northem杂交相结合的方法研究拟南芥不同生态型接种马铃薯晚疫病菌后抗病相关基因的表达，结果表明，Bay一O生态型经过sA处理后接种马铃薯晚疫病菌，至少诱导了3个抗病相关基因(Ai2932680、Atlg67980、PR一l)的表达，Col生态型经JA处理后，诱导了PDFI，2基因的表达，Bay一。生态型经JA处理后，诱导了PR一4基因的表达，Est一1及Col生态型经SA处理后，诱导了PR一3基因的表达。更多还原

【Abstract】 Resistance of 12 different Arabidopsis ecotypes and genetypes to some important fungal pathogens were studied, and different ecotypes which were different resistance to the same pathogen had been obtained. The molecular mechanisms of resistance and expression of defense-related genes after inoculation were also studied in this paper.All the 12 Arabidopsis ecotypes tested were incompatible to Fusarium campestris, F. oxysporum f.sp.niveum, Setosphaeria turcica and Erysiphe cichoracearum. Some ecotypes were compatible to Alternaria brassicicola, Botrytis cinerea (two strains), Phytophthora infestans and Peronospora parasitica. Among them Col was compatible to B. cinerea and P. parasitica, C24 was compatible to P. parasitica, NahG was compatible to B. cinerea. Est-1, Bay-0 were incompatible to A. brassicicola, B. cinerea, P. infestans and P. parasitica. The application of SA or JA to Est-1 and Bay-0 plants enhanced resistance to P. infestans.To study roles of defensive enzymes in the host resistance, their activities were analyzed in inoculated and uninoculated plants. The results indicated that PAL, POD and PPO activities were induced in inoculated plants, the resistant ecotype Col usually exhibited a quicker and higher increase in the activity of these enzymes than susceptible ecotypes. The changes of SOD activity followed the process: the activity of resistant ecotype Col showed first a little increase then a slow decrease and another swift increase, reach the highest value in the seventh day after inoculation, and finally decreased again in the ninth day after inoculation. As compared with susceptible ecotypes Est-1 and Bay-0, SOD activity increased quickly in seven days after inoculation and dropped down in the ninth day after inoculation. The CAT activity of Col, Est-1 and Bay-0 ecotypes also showed a change after infection with P. infestans spores. The CAT activity in Col kept increasing until reaching the highest level in the seventh day and then dropped slowly. As to CAT activity in ecotypes Est-1 and Bay-0, there were no any change during the first day after inoculation but increased gradually after and reached the maximum level after 3-5days. The maximum value of CAT in the resistant ecotype Col was higher than in the susceptible ecotypes Est-1 and Bay-0.Other non-enzyme substances which related to resistance were also observed after inoculation with P. infestans. It was found that : (1)The change of susceptible host cell membrane permeability occurred usually earlier and a wider range compared with resistant ecotypes. (2) MDA which is generated from peroxidized membrane lipid, increased in all three ecotypes used. The increase of MDA in Col after inoculation occurred usually earlier but the maximum value was lower while compared with Est-1 and Bay-0 ecotypes.DDRT-PCR method was optimal to study the differential gene expression between inoculated and uninoculated plants of Col, Est-1 and Bay-0, and total 108 differentially expressed cDNA fragments were found. Among them 57 cDNA fragments were found to hybridize to cDNA from inoculation and uninoculation plants. Four of the differentially expressed cDNA fragments were cloned and sequenced. Their lengths were 269bp and 466bp, respectively. The fragment 7-6 (466bp) was derived from gene which encodes a member of the 1-aminocyclopropane-l-carboxylate synthase. 6-6(269bp) was derived from gene At5g32405.1 which the function was unknown.Reverse Northern blotting method was exploited to study the expression of the pathogen-induced resistant genes. It was shown that the product of second PCR amplification of Col plants displayed a positive reaction to PR-3. Bay-0 displayed positive reaction to PR-1. Est-1 displayed positive reaction to At2g32680, At1g67980, PDF 1.2, PR-3 and PR-4. Both reverse Northern blotting and DDRT-PCR were used to study the expression of defense-related genes in inoculated and uninoculated Arabidopsis plants. SA induced expression of At2g32680, Atlg67980 and PR-1 in Bay-0 plants. PDF1.2 was induced after treatment by JA in Col plan更多还原