【作者】 刘新颖；

【导师】 康振生；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2010， 硕士

【摘要】 由条形柄锈菌(Puccinia striiformis f. sp. tritici)引起的小麦条锈病是世界各国范围内普遍发生的小麦病害之一。小麦条锈病的大面积流行可造成小麦严重减产,选育和利用抗锈品种是控制小麦条锈病最为可靠的方法。过敏性坏死反应是小麦受条锈菌侵染后的典型抗病反应,通过研究自噬基因在HR-PCD中的作用与分子机制,以揭示小麦与条锈菌互作的致病机理。本研究通过分析拟南芥、水稻以及短柄草的自噬基因序列,根据保守区域设计引物,成功克隆了六个小麦白噬相关基因,分别命名为TaATG8a、TaATG8c、TaATG8h、TaATG18c、TaATG3和TaATG6。TaATG8a长695 bp,编码119个氨基酸,TaATG8c长647 bp,编码119个氨基酸,TaATG8h长532 bp,编码117个氨基酸,序列分析表明编码蛋白都具有一个类泛素折叠的保守结构。TaATG8a、TaATG8h与拟南芥的ATG8蛋白关系最近；TaATG8c与玉米的ATG8蛋白关系最近。三个小麦Atg8基因均不同程度受条锈菌诱导,接种条锈菌后TaATG8a在非亲和组合中上调表达在,在亲和组合中下调表达；TaATG8c和TaATG8h非亲和与亲和组合中都上调表达。TaATG8a和TaATG8h受脱落酸、乙烯和茉莉酸甲酯诱导上调表达,水杨酸诱导其下调表达。TaATG8c对外源激素不敏感,处理后轻微上下浮动。TaATG8a和TaATG8h可能通过茉莉酸和乙烯信号途径介导小麦对条锈菌的防御反应,水杨酸与茉莉酸在小麦抗条锈防御反应中存在明显的拮抗作用。在环境胁迫条件下,TaATG8a受干旱、高盐和机械伤害诱导,低温环境下则下调表达；在干旱和高盐环境中,TaATG8c下调表达,在机械伤害和低温环境中TaATG8c上调表达；TaATG8h对环境胁迫十分敏感,在所有处理中都上调表达,并且表达量很高。可见小麦中的Atg8成员可能分别或共同参与了不同的抗逆机制。TaATG18c长1205 bp,编码348个氨基酸,分析发现TaATG18c含有多个WD40重复序列,并且含有三个跨膜结构域,与拟南芥亲缘关系最近。定量分析表明,TaATG18c在亲和互作组合中,上调表达；TaATG18c受脱落酸诱导上调表达,受乙烯、茉莉酸甲酯和水杨酸诱导其下调表达；在干旱、高盐和机械伤害条件下TaATG18c表达量下降：低温环境下TaATG18c上调表达。条锈菌侵染小麦后,TaATG18c有可能参与了小麦体内营养物质的转运。TaATG3长954 bp,编码317个氨基酸；TaATG6长1556 bp,编码504个氨基酸,编码蛋白都存在一个跨膜螺旋区域TaATG3编码蛋白与大麦ATG3亲缘关系最近,与水稻亲缘关系次之；TaATG3编码蛋白与水稻ATG6亲缘关系最近,与大麦等禾本科作物次之。定量研究表明,TaATG3和TaATG6基因的转录表达受小麦条锈菌的诱导,并且非亲和组合表达量远远高于亲和组合,它们可能参与了小麦与条锈菌的非亲和互作。通过VIGS技术沉默小麦TaATG3和TaATG6基因发现,小麦叶片过敏性坏死斑面积明显增大,出现大面积的褪绿,死亡扩散到健康的叶肉组织。细胞荧光染色观察表明,转染BSMV-ATG3、BSMV-ATG6植株的叶片侵染点的坏死细胞数目增多,坏死面积扩大,条锈菌侵染菌丝长度比对照有所增加,条锈菌的扩展情况更加严重。TaATG3和TaATG6基因参与了过敏性坏死反应中抑制细胞死亡扩展的进程,但与寄主细胞死亡起始无关,并对小麦抗病性,抑制病原菌扩展起到了一定程度的作用。更多还原

【Abstract】 Wheat stripe rust, caused by Puccinia striiformis f.sp. tritici, is widely spread around the world. The pandemic of wheat stripe rust can cause severe yield reduction of wheat, selection and use of rust-resistant varieties are the most reliable method in controlling the disease. Hypersensitive response is a typical resistance reaction,of wheat after infected by stripe rust, studying the role and molecular mechanism of autophagy genes in HR-PCD is very important to reveal the interaction between wheat and stripe rust pathogenic mechanism.In this study, the analysis of Arabidopsis, Oryza Sativa and Brachypodium sylvaticum autophagy gene sequence, primers were designed according to conserved regions, the successful cloning of the six wheat autophagy-related gene, named TaATG8a, TaATG8c, TaATG8h, TaATG18c, TaATG3 and TaATG6.TaATG8a length 695bp, encoding 119 amino acids, TaATG8c length 647bp, encoding 119 amino acids, TaATG8h length 532bp, encoding 117 amino acid, sequence analysis showed that three encoded proteins has a class of conserved ubiquitin fold.TaATG8a and TaATG8h sharing high homology with Arabidopsis ATG8 protein as well as TaATG8c sharing high homology with corn ATG8 protein respectively.Three wheat Atg8 genes were induced by stripe rust in different levels. After inoculation of stripe rust TaATG8a expression was up-regulated in incompatible interaction and down-regulated compatible interaction. TaATG8c and TaATG8h expressions were both up-regulated in incompatible and compatible interactions. TaATG8a and TaATG8h transcripts in wheat leaves was induced by exogenous applied methyl jasmonate (MeJA), ABA and ethylene. However, salicylic acid (SA) had no obvious effect on the expressions. TaATG8c transcript is not sensitive to exogenous hormones, processed slightly fluctuate. TaATG8a and TaATG8h possible through mediated wheat stripe rust by the JA and ethylene signaling pathways in the defense response, salicylic acid and jasmonic acid are obvious antagonism in defense response of wheat stripe rust. Environmental stimuli, including drought, high salinity and wounding, also induced TaATG8a expression.while low-temperature lead reducesd TaATG8a expression.Under drought and high salinity environment, TaATG8c expression was down-regulated, while under wounding and low-temperature environment the expression of TaATG8c was up-regulated.; TaATG8h was very sensitive to environmental stress and always up-regulated in all treatments. The Atg8 members may separately or jointly involved in the different resilience mechanisms of wheat.TaATG18c length 1205bp, encoding 348 amino acids, sequence analysis showed that TaATG18c has multiple WD40 repeats sequences and contains three transmembrane domains.TaATG18c sharing high homology with Arabidopsis ATG18. Quantitative analysis showed that, TaATG18c transcripts was induced in compatible interaction. TaATG18c transcripts in wheat leaves was induced by exogenous applied methyl jasmonate (MeJA), ABA and ethylene. However, salicylic acid (SA) had no obvious effect. Environmental stimuli, including drought, high salinity and wounding, also induced TaATG18c expression.while low-temperature lead reducesd TaATG18c expression.TaATG18c may be involved in the nutrients transfer in wheat after the wheat stripe rust infection.TaATG3 length 954bp, encoding 317 amino acids; TaATG6 length 1556bp, encoding 504 amino acids, two encoded proteins both has a transmembrane helix region. TaATG3 sharing high homology with barley ATG3 and TaATG6 sharing high homology with rice ATG6. Quantitative result shows that, TaATG3 and TaATG6 gene transcription induced by wheat stripe rust, and the expression of incompatible interaction is much higher than the compatible interaction, they may be involved in the interaction between wheat and the stripe rust. After silencing wheat TaATG3 and TaATG6 genes by VIGS technology, it was found that of area of chlorosis was larger in wheat leaves and cell death had spread to healthy leaf tissue. Fluorescence staining showed increased number of dead cells points of transfected BSMV-ATG3 and BSMV-ATG6 leaves, also the necrosis area had expand, the length of stripe rust infection hyphae had slight increased, the expansion of stripe rust even more serious. TaATG3 and TaATG6 genes are likely involved in the hypersensitive cell death response in the inhibition of the process of pothogen expansion, and wheat disease resistance.更多还原