【作者】 王晓敏；

【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， 博士

【摘要】 小麦条锈病是由条形柄锈菌小麦专化型（Puccinia striiformis Westend f. sp. tritici Eriks. & Henn.）引起的一种重要的小麦（Triticum aestivum L.）气传叶部病害,也是我国乃至世界上所有产麦国家危害最严重的世界性小麦病害之一。以多年来国内外专家学者的研究和生产实践证明,合理利用抗病基因,培育和推广高效、稳定、广谱、持久的抗病品种是控制与治理小麦条锈病最经济、安全、有效的途径。优良的抗病种质与基因资源是产生突破性抗病育种的物质基础;深入研究小麦的抗病机制,是抗病育种的必由之路;加强对小麦与条锈菌互作过程中的组织学、组织化学和分子生物学方面的研究,为阐明小麦与条锈菌互作的分子机制奠定了坚实的基础,对抗病防治和抗病遗传育种工作具有重要的理论和指导意义。本文一方面,采用光学显微镜和组织化学研究方法,通过比较由携带Yr10的抗病小麦品种Moro和感病品种Fielder与条锈菌组成的非亲和与亲和体系互作反应的不同,阐明病原菌的发展、寄主的过敏性反应和活性氧（ROS）的积累在细胞学上的关系;并利用实时定量PCR （qRT-PCR）,对一系列防御相关基因在非亲和与亲和组合中进行表达谱的研究,将细胞学反应与基因表达调控相结合,对Yr10介导的对条锈菌的防御反应中涉及的关键因素进行揭示。另一方面,本文从实验室前期以小麦品种水源11与条锈菌生理小种CYR23为材料,利用抑制性差减杂交技术已经构建完成的非亲和条锈菌诱导的小麦叶片cDNA文库或NCBI小麦EST数据库中,共挑选了3个候选基因。通过电子克隆结合RT-PCR技术,克隆得到这3个基因全长序列,它们分别为:小麦含CBS结构域的蛋白基因、小麦EF-手型钙结合蛋白基因和小麦受翻译调节的肿瘤蛋白基因;通过生物信息学比对和分析了解这些基因的基本特征;利用qRT-PCR技术,分析这些基因在对条锈菌侵染的防御反应、非生物胁迫和环境影响等过程中的分子特征和表达谱;利用基因枪转化法在洋葱表皮细胞对小麦EF-手型钙结合蛋白基因和小麦受翻译调节的肿瘤蛋白基因进行瞬时表达,明确其亚细胞定位;运用BSMV-VIGS技术对小麦受翻译调节的肿瘤蛋白基因进行功能分析。通过这些实验,初步明确这3个基因在小麦与条锈菌互作体系中的分子生物学功能。具体研究内容和结果如下:1.在小麦抗病品种Moro （携带Yr10抗条锈病基因）和感病品种Fielder中,至接种后6～12 d,条锈菌在叶片中的侵入和定殖非常相似。但是在此之后,Moro发生活性氧迸发和过敏性坏死反应阻止了病原菌的进一步扩展。在非亲和组合中,防御信号分子基因于接种后较早期2～6 d特异上调表达,病程相关蛋白（Pathogenesis-related Protein,PR-protein）基因则于接种后4～14 d有特异的上调表达。结果表明识别反应发生在寄主与病原菌互作早期,而阻止病原菌发展的关键的PR-蛋白介导的防御反应发生在互作后期。本研究是第一次在谷类锈菌中将详细的形态学方面的防御反应,包括活性氧迸发和HR,与已经注释的防御基因的表达谱相结合,以目前的寄主与病原菌互作模式阐明了Yr10和条锈菌的关系。2.小麦含CBS结构域蛋白的基因TaCDCP1 （Triticum aestivum CBS domain containing protein 1）,开放阅读框654 bp,编码217个氨基酸;TaCDCP1拟编码的蛋白预测具有两个典型的CBS保守结构域,不含跨膜区,无信号肽,定位在叶绿体基质内;经过同源比对,TaCDCP1氨基酸序列与大麦、水稻和玉米等的同源序列的相似性较高;该基因在小麦叶中的表达量显著高于在根和茎中的;在小麦与条锈菌的非亲和与亲和组合中,TaCDCP1基因均受到条锈菌诱导,非亲和组合表达量在侵染前期（接种后18～48 h）高于亲和组合,而在侵染后期（接种后96～120 h）低于亲和组合;外源植物激素脱落酸诱导该基因上调表达,苄基腺嘌呤、乙烯、赤霉素、茉莉酸甲酯和水杨酸处理后,其表达量在不同程度上受到抑制;TaCDCP1在低温和干旱条件下表达量上调,经机械伤害和高盐处理后表达量无明显差异。以上表明TaCDCP1可能通过脱落酸等信号途径参与小麦对条锈菌的防御反应,同时参与低温和干旱环境下的防御反应。结果对于明确CBS结构域的功能以及CBS结构域蛋白尤其是TaCDCP1在小麦与条锈菌互作中的作用奠定了基础。3.小麦EF-手型钙结合蛋白基因TaCab1 （Triticum aestivum calcium binding EF-hand protein 1）,DNA序列没有内含子,开放阅读框651 bp,编码216个氨基酸;TaCab1拟编码的蛋白预测具有一个信号肽,一个跨膜区域,一个油体钙蛋白保守结构域（caleosin conserved domain）和一个单独的EF-手型基序;TaCab1与大麦中EF-手型钙结合蛋白基因BCI-4编码的蛋白同源性高达92 %;洋葱表皮细胞的瞬时表达结果显示TaCab1基因编码一个跨膜蛋白;TaCab1的表达可能受到钙离子浓度的调控;该基因在小麦叶中的表达量显著高于在根和茎中;尽管在非亲和与亲和组合中表达整体表现为相似的上调趋势,TaCab1在亲和组合中的表达量显著高于在非亲和组合中的;在水杨酸处理后2～24 h,TaCab1的表达整体保持上调,在其他不同激素和非生物胁迫处理下,TaCab1基因于处理早期被诱导,且均有不同程度的上调表达,但均无水杨酸处理后上调幅度大。以上表明,TaCab1可能通过水杨酸信号途径参与小麦对条锈菌的防御反应负调控,同时参与对环境压力的基础抗性反应。这些结果揭示了TaCab1在小麦应对生物与非生物胁迫和条锈菌致病过程中发挥的作用,为进一步研究TaCab1在小麦与条锈菌互作中的特殊功能奠定基础。4.小麦受翻译调节的肿瘤蛋白基因TaTCTP1 （Triticum aestivum translationally controlled tumor protein 1）,该基因DNA序列全长1647bp,包含4个内含子序列和5个外显子序列;该基因开放阅读框为507 bp,编码168个氨基酸,预测其不含跨膜区,无信号肽;具有Mss4-like和Mss4/TCTP-associated超家族保守结构域以及TCTP₁和TCTP₂两个TCTP保守结构特征区;TaTCTP1与小麦受翻译调节的肿瘤蛋白TaTCTP序列（GenBank登录号为AAM34280）同源性高达98 %;亚细胞定位结果表明TaTCTP1编码一个胞浆蛋白;该基因的表达可能受到钙离子浓度的调控;该基因在小麦根、茎和叶中表达水平一致;TaTCTP1受小麦条锈菌诱导表达,非亲和组合表达量在侵染前期（接种后12～48 h）高于亲和组合,于接种后18 h在非亲和组合中出现第一个表达高峰,而在侵染后期（接种后96～120 h）,虽然非亲和组合中TaTCTP1出现第二个表达高峰,但在亲和组合中TaTCTP1有较高的相对表达量;TaTCTP1基因仅在乙烯、高盐和低温处理早期被诱导表达,对于其他激素包括水杨酸、茉莉酸等的处理,干旱和伤害处理后,其表达量基本无变化。利用BSMV-VIGS分析,TaTCTP1沉默后的水源11植株在挑战接种CYR23后,小麦叶片由原来的抗病反应型变为感病反应型,产生大量孢子;在挑战接种CYR31后,小麦叶片的症状与对照的反应型一致。以上结果表明,TaTCTP1可能通过乙烯信号途径参与对低温和高盐等逆境的抗性反应,并在小麦对条锈菌的抗病途径中发挥着十分重要的作用,这为进一步研究TaTCTP1在小麦与条锈菌互作中的特殊功能奠定基础。更多还原

【Abstract】 Stripe rust, caused by Puccinia striiformis Westend f. sp. tritici Eriks. & Henn. （Pst）, is one of the most widely destructive leaf diseases of wheat （Triticum aestivum L.） in the world. Wheat yield can be greatly reduced, even completely destroyed depending upon the level of the disease epidemic. It has been proven that breeding and rational utilization of disease-resistant varieties is the safest, most economical and effective method to control wheat stripe rust. Investigations to the resistant mechanism of the host plant and the interactions between wheat and the stripe rust pathogen from histological, histochemical and molecular biological aspects are important to give further information for the rational use of resistant genes in the improvement of resistance in cultivars.This study can be divided into two parts, one part is employed light and fluorescent microscopy, H2O2 staining, and defense gene profiling to compare the incompatible interaction involving resistance gene Yr10 and Pst in Moro wheat with a compatible interaction involving Fielder in order to elucidate the relationships among morphological aspects of pathogen development, the HR and ROS. The expression profiles of a series of defense-related genes which were studied by qRT-PCR permitted development of an integrated, time-course portrait of the host-parasite interaction of Pst in susceptible Fielder and Yr10-resistant Moro linking morphological responses to regulation of gene expression.The other part, on the basis of previous constructed suppression subtractive hybridization （SSH） the incompatible cDNA library of wheat （cv. Suwon 11） leaves infected by Pst CYR23, or from the NCBI GenBank wheat EST database, we isolated and characterized 3 full length cDNA sequences which were the CBS domain containing protein gene, the calcium binding EF-hand protein gene and translationally controlled tumor protein gene from wheat leaves infected with Pst through in silico cloning and reverse transcription PCR （RT-PCR） approaches; Characterized the molecular features and transcription profiles of these genes in the wheat defense responses to Pst, phytohormones and abiotic stress stimuli by qRT-PCR analysis; Subcellular localization of the calcium binding EF-hand protein gene and the translationally controlled tumor protein gene by introducing the GFP fusion protein constructs via particle bombardment in onion epidermal cells; Functional anlysis of the translationally controlled tumor protein gene was carried out thought BSMV-VIGS （Barley Stripe Mosaic Virus-Virus Induced Gene Silencing） method. The main studies contents and results are as follows:1. This study integrated defense-related genes profiling with histological and histochemical studies to develop a detailed time-course description of Pst penetration and infection of susceptible Fielder wheat and resistant Moro that contains the Yr10 gene for stripe rust resistance. Penetration and establishment events of the fungus within the leaf are very similar in both Fielder and Moro until approximately 6 to 12 days after inoculation （dai） after which phenomena associated with oxidative burst and hypersensitive response （HR） were observed in Moro that terminated further growth of the pathogen. Differential upregulation of transcripts of defense signaling genes in Moro compared to Fielder were observed as early as 2-6 dai and from 4-14 dai among defense-related PR-proteins. We demonstrated that recognition responses in hosts occur early in the host-parasite interaction but that key defense responses that terminate pathogen development and response occurred later.These results are the first among cereal rusts to integrate detailed morphological aspects of defense reactions including oxidative burst and HR with transcript profiling of annotated genes and permitted interpretation of the Yr10-stripe rust interaction in the context of current models of host-parasite interactions. These results will also permit identification of unannotated genes based on profile expression in the near future and thus uncovering additional key factors involved in the Yr10 mediated resistance response to P. striiformis in wheat.2. The CBS domain containing protein gene, tentatively designated as TaCDCP1 （Triticum aestivum CBS domain containing protein 1）, was predicted to encode 217 amino acids protein which contained two conserved cystathionine beta-synthase （CBS） domains and was without transmembrane domain or signal peptide sequence. The deduced protein was predicted existing in chloroplast stroma. The amino acid sequence of TaCDCP1 shares 92%, 72% and 63% identify with the homologs in barley （Hordeum vulgare） , rice （Oryza sativa） and maize （Zea mays）, respectively. The TaCDCP1 gene was highly expressed in leaves than in roots and stems. Challenged by Pst, TaCDCP1 was induced by this fungus in both incompatible and compatible interactions, with the maximal expression at 18 h post inoculation （hpi） and 96 hpi, respectively. Its transcript accumulation was much higher in the incompatible interaction than in the compatible interaction at the early stage of infection （18-48 hpi）, but much lower at the late stage （96-120 hpi）. The expression of TaCDCP1 was also up-regulated after treated by phytohormones such as abscisic acid （ABA）, and down-regulated by benzyladenine, ethylene, gibberellins, methyl jasmonate and salicylic acid to a certain degree. And it was obviously up-regulated by various abiotic stresses, such as low temperature and drought. However, mechanical wound and high salinity stress could not induce the expression of TaCDCP1. These results suggest that TaCDCP1 is probably involved in the disease resistance and defense response in wheat to Pst through ABA pathways, and also participate in the signal transmission pathways under low temperature, and drought conditions.3. The calcium binding EF-hand protein gene, designated as TaCab1 （Triticum aestivum calcium binding EF-hand protein 1）, did not have an intron and was predicted to encode a 216 amino acid protein which possesses an N-terminal region with a signal peptide, a transmembrane domain, an EF-hand motif and a caleosin domain. TaCab1 The results of transient assays with constructs of TaCab1 with green fluorescent protein （GFP） gene indicated that TaCab1 encodes a transmembrane protein. Quantitative real-time PCR （qRT-PCR） analyses revealed that TaCab1 was highly expressed in leaves than roots and stems. Although up-regulated expression profiles of TaCab1 were quite similar in both incompatible and compatible interactions, its transcript accumulation in the compatible interaction was much higher than in the incompatible interaction. The transcription of TaCab1 was consistently up-regulated after treated with salicylic acid （SA）, and it also up-regulated at different degrees after treated by other phytohormones and stress stimuli. These results suggest that TaCab1 is involved in the plant-pathogen recognition, symptom development, and the basal tolerance to biotic and abiotic stresses through the SA signaling pathway.4. The wheat translationally controlled tumor protein designated as TaTCTP1 （Triticum aestivum translationally controlled tumor protein 1）. It had 1647 bp DNA sequence with 4 introns and 5 extrons. TaTCTP1 was predicted to encode a 168 amino acid protein, which possesses an Mss4-like and Mss4/TCTP-associated superfamily conserved domains and 2 TCTP conserved sites, TCTP₁ and TCTP₂, and was without transmembrane domain or signal peptide sequence. The results of transient assays indicated that TaTCTP1 encodes a cytoplasmic protein. qRT-PCR analyses revealed that TaTCTP1 was constitutively expressed in leaves, roots and stems. The transcription level of TaTCTP1 was much higher in the incompatible interaction than in the compatible interaction at the early stage of infection （12-48 hpi）, a little lower at the late stage （96-120 hpi）, but was still at a high level. The transcription of TaTCTP1 was up-regulated after treated by ethylene, low temperature and high salinity. Functional analysis system mediated by BSMV-VIGS revealed that wheat plants with silenced gene of TaTCTP1 produced considerable amount of sporulation after inoculated with Pst race CYR23, and the leaf symptom was not changed after inoculated with Pst race CYR31. These results suggested TaTCTP1 could play an very important role in wheat resistant response against Pst and defense response to low temperature and high salinity through ethylene pathway.更多还原