【作者】 李娟；

【导师】 王保通；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2019， 硕士

【摘要】 条锈病是世界上多个国家和地区普遍发生的小麦病害之一。由于条锈菌毒性变异快和单一抗病品种的大面积种植,致使抗病品种抗性很容易丧失。防治该病最经济、有效和安全的措施是培育和利用抗病品种,而优异的小麦条锈病抗源及抗病基因是小麦抗病育种的基础。普通小麦-滨麦草衍生系M8664-3对中国小麦条锈菌多个主要流行小种具有全生育期抗性,尤其对条锈菌CYR33在苗期会产生过敏性坏死反应。本研究在前期已经将M8664-3对CYR33的抗病基因Yr M8664-3定位于小麦4AL染色体的基础上,进行Yr M8664-3定位区间内抗病相关基因的筛选及其功能验证,主要取得如下研究结果:1.M8664-3接种条锈菌CYR33后产生了明显的坏死斑。组织病理学观察表明,M8664-3显著抑制了CYR33的生长发育,产生了强烈的活性氧迸发和过敏性坏死反应。将Yr M8664-3的定位区间与中国春全基因组序列对比分析,得到有类似抗病结构域的基因19个。对这19个基因进行初步实时荧光定量分析,选择表达量明显上升和明显下降的基因进行大麦花叶病毒诱导的基因沉默(BSMV-VIGS)功能验证,发现Ta＿Pes＿BRCT(暂命名)和Ta LRP(暂命名)两个基因与小麦抗条锈病相关。2.Ta＿Pes＿BRCT的序列分析表明,Ta＿Pes＿BRCT编码一个具有Pescadillo＿N和BRCT两个结构域的蛋白,这两个结构域都与DNA损伤修复和损伤应答有关。定量分析表明,Ta＿Pes＿BRCT在M8664-3接种CYR33后表达量下降。BSMV-VIGS验证结果表明,Ta＿Pes＿BRCT沉默之后的植株叶片上产生了明显的孢子堆。组织病理学观察结果显示Ta＿Pes＿BRCT沉默后对条锈菌生长的抑制作用明显减弱。Ta＿Pes＿BRCT在高温(37℃)处理下表达受到抑制,在模拟干旱(PEG6000)处理下被诱导表达;说明Ta＿Pes＿BRCT的抗病反应有可能参与了植物抗旱和高温调控的过程。3.Ta LRP的序列分析表明,Ta LRP编码一个有连续三段亮氨酸重复序列的跨膜蛋白。定量分析表明,Ta LRP在M8664-3接种CYR33后表达量上升。BSMV-VIGS验证结果表明,Ta LRP沉默之后的植株叶片上产生了明显的孢子堆。组织病理学观察结果显示Ta LRP沉默后对条锈菌生长的抑制作用明显减弱。在茉莉酸(JA)处理下Ta LRP表达受到抑制,而在乙烯(ET)和模拟干旱(PEG6000)模拟干旱处理下被诱导表达,说明Ta LRP的抗病反应可能与ET和JA代谢通路相关,也可能参与植物抗旱的过程。更多还原

【Abstract】 Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most common and severity wheat diseases in many countries and regions in the world.Due to the rapid virulence variation of Puccinia striiformis f.sp.tritici and large-scale planting of single disease-resistant varieties,resistance of these varieties is easily overcome by new Pst races.Cultivate and utilization of resistant varieties is the most economical,effective and safe measure to control this disease,and the excellent wheat stripe rust resistance resources and disease resistance genes are the basis for wheat disease-resistant breeding.The wheat-Leymus mollis introgression line M8664-3 exhibits all-stage resistance to Chinese Pst races,especially showed obvious necrosis when infected with Pst CYR33 at the seedling stage.In our previous study,a single dominant gene Yr M8664-3 conferring resistance to Pst CYR33 was identified and located on wheat chromosome 4AL.In this study,the sequences of the flanking markers of Yr M8664-3 were blasted against the genome sequence of T.aestivum cv.Chinese Spring and therelated genes for disease resistance and its functional verification were identified and verified,the main results was as follows:1.M8664-3 produced significant necrotic plaques when inoculated with CYR33.Histopathological observation showed that the expend of CYR33 in M8664-3 was significantly inhibited,and M8664-3 produced strong reactive oxygen burst and hypersensitive reaction.After comparied with the Chinese spring genome sequence,19 resistance related genes with the similar disease resistance domains were obtained.These 19 genes were screened by real-time quantitative PCR analysis(q RT-PCR).The 19 genes with significantly up-regulated and down-regulated expression were selected and used for functional verification of barley stripe mosaic virus-induced gene silencing(BSMV-VIGS).The results indicated that Ta＿Pes＿BRCT(temporary nomenclature)and Ta LRP(temporary nomenclature)are associated with wheat resistance to stripe rust.2.Sequence analysis of Ta＿Pes＿BRCT indicated that Ta＿Pes＿BRCT encodes a protein with two domains,Pescadillo＿N and BRCT,both of which are involved in DNA damage repair and damage response.q RT-PCR analysis showed that Ta＿Pes＿BRCT is a down-regulated gene when M8664-3 inoculated with CYR33.Silencing Ta＿Pes＿BRCT led to enhanced susceptibility to Pst(in terms of the significant uredinial pustules was produced on the leaves of plants)than non-silenced.Histopathological observation showed that the inhibition of growth of stripe rust was significantly attenuated after Ta＿Pes＿BRCT silencing.The expression of Ta＿Pes＿BRCT was inhibited under high temperature(37°C)treatment and induced under simulated drought(PEG6000)treatment.It indicated that the resistance reaction of Ta＿Pes＿BRCT may be involved in the process of plant drought resistance and high temperature regulation.3.Sequence analysis of Ta LRP revealed that Ta LRP is a transmembrane protein with three consecutive leucine repeats.Real-time quantitative PCR analysis showed,Ta LRP is an up-regulated gene when M8664-3 infected with the CYR33.Silencing Ta LRP led to enhanced susceptibility to Pst(in terms of the significant uredinial pustules was produced on the leaves of plants)than non-silenced.Histopathological observation showed that the inhibition of growth of stripe rust was significantly attenuated after Ta LRP silencing.The expression of Ta LRP was inhibited by jasmonate(JA),but induced by ethylene(ET)and simulated drought(PEG6000)simulated drought,indicating that the resistance of Ta LRP may be related to ET and JA metabolic pathway and may also be involved in plant drought and salt tolerance processes.更多还原