【作者】 薛仁风；

【导师】 王述民； 朱振东；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2012， 博士

【摘要】 普通菜豆是人类主要食用豆类作物之一，营养价值高，栽培面积大。但在菜豆栽培生产中，尖镰孢菌枯萎病一直是菜豆生产中严重的病害，给菜豆生产带来较大损失。因此，筛选具有稳定抗性的菜豆种质资源，揭示普通菜豆镰孢菌枯萎病的抗病机理，发掘并利用优异的抗病基因，改良普通菜豆抗病性，对保障我国食物安全、提高菜豆生产的经济效益具有重要的理论和现实意义。本研究根据普通菜豆枯萎病原菌（Fusarium oxysporum f. sp. phaseoli）中与其致病力密切相关的一个转录因子基因（ftf1基因）设计了一对特异性引物，结合荧光定量PCR技术能够准确区分抗病性不同的菜豆种质，通过cDNA-AFLP技术揭示了普通菜豆与病原菌互作过程中的差异表达基因，克隆了2个寄主防御反应相关基因，钙调素基因PvCaM1和过氧化物酶基因PvPOX1，并通过外源激素诱导的方法探索菜豆抗病信号传导途径及其抗病机理，以期为普通菜豆抗镰孢菌枯萎病抗性遗传改良提供基因资源和理论依据。获得如下结果：1.从全国6个省的8个地点采集的病株上分离病原菌，通过形态学鉴定得到59株尖镰孢菌，并从致病性和分子生物学方面鉴定出致病菌37株。根据F. oxysporum f. sp. phaseoli中ftf1基因设计了一对特异性引物QFopA/QFopB，该引物组合只能在F. oxysporum f. sp. phaseoli中扩增到一个149bp的片段，因此可以用于准确鉴定菜豆枯萎病原菌，从而为定量地筛选抗性较好的菜豆种质资源奠定了基础。2.利用枯萎病原菌FOP-DM01菌株，比较了玉米粉接种体法、下胚轴双孔注射法及接种体蘸根法的接种效果，选用玉米粉接种体法从362份普通菜豆种质资源中鉴定出抗病种质16份，其中2份抗性较好，品种名称分别为260205和黑芸豆，统一编号分别为：F0005035和F0004851。利用F. oxysporum f. sp. phaseoli特异性引物QFopA/QFopB并结合荧光定量PCR技术，最低能够检测到1pg的病原菌DNA，通过对感病菜豆品种BRB-130，A0640-1和抗病菜豆品种260205，黑芸豆根和茎中病原菌的定殖量进行分析，结果表明，接种病原菌6d后，BRB-130和A0640-1根中的定殖量均显著高于病原菌在260205和黑芸豆中的定殖量。3.通过cDNA-AFLP技术揭示了感病材料BRB-130和抗病材料260205分别与FOP-DM01菌株互作中差异表达基因，共获得443个差异表达片段，其中172个已经测序，测序的片段其中包括许多植物抗病相关基因，如：生长素调节蛋白（GH3auxin-regulated protein）、钙调素蛋白（calmodulin）、过氧化物酶（peroxidase）等，并通过荧光定量PCR技术验证差异表达基因在互作过程中表达量的变化；扫描电镜（Scanning electron micrographs, SEM）和透射电镜（Transmissionelectron micrographs, TEM）观察结果显示，BRB-130根和茎的组织结构和细胞结构被病原菌严重破坏，而260205根和茎被病原菌浸染后组织结构基本正常；260205被病原菌侵染后根和茎中苯丙氨酸解氨酶（Phenylalanine ammonia lyase, PAL）和过氧化物酶（Peroxidase, POX）的活性要显著高于BRB-130，表明这两种植物防御相关酶在菜豆抗病反应中发挥重要的作用。此外对菜豆根和茎中H₂O₂和O₂^-的含量进行了测定，结果表明，260205被病原菌侵染后根和茎中H₂O₂和O₂^-的含量均高于BRB-130，说明活性氧（ROS）在菜豆抗镰饱菌枯萎病抗性反应中同样具有重要的作用。4.利用表达序列标签（EST）克隆了含有编码普通菜豆CaM基因，命名为PvCaM1（JN418801），同源分析结果显示，PvCaM1基因与百脉根、西瓜的CaM基因亲缘关系最近，分别达到77%和76%。荧光定量PCR分析表明，PvCaM1基因受FOP-DM01菌株诱导表达，而且叶中的表达量高于根和茎中的表达量。PvCaM1基因表达量也受外源植物激素脱落酸（ABA）、茉莉酸甲酯（MeJA）和乙烯利（ETH）诱导上调，在根、茎、叶中均有不同程度的表达，其中叶中表达量最高。5.利用EST克隆了含有编码普通菜豆POX基因，命名为PvPOX1（JQ627838），同源分析结果显示，PvPOX1与大豆POX基因的亲缘关系最近，达到90%，与苜蓿次之，达85%。荧光定量PCR分析表明，在根和茎中PvPOX1基因均受FOP-DM01菌株显著诱导表达，并且伴随着POX活性的显著增强及H₂O₂含量的升高，PvPOX1基因表达量的升高与POX活性及H₂O₂含量的变化具有明显的相关性。PvPOX1基因在根、茎、叶、花和荚中均有不同程度的表达，叶中最低，荚中最高。PvPOX1基因表达量也受水杨酸（SA）和ABA等植物激素及机械损伤、盐、干旱等非生物胁迫诱导表达。6.采用SA、MeJA、ETH、多效唑（Paclobutrazol）和去甲二氢愈创木酸（NDGA）等化学药品处理菜豆BRB-130，结果表明SA处理菜豆叶片使植株根中内源SA的含量升高，并显著提高植株对枯萎病原菌FOP-DM01菌株的抗病性。此外，SA能够显著地诱导菜豆SAR反应，从而降低了FOP-DM01菌株在菜豆根中增殖能力，体外实验结果表明，SA对FOP-DM01菌株并没有直接的抑菌活性；另外，SA诱导菜豆根组织中PAL和POX活性和H₂O₂和O₂^-的含量显著升高，从而诱导菜豆产生HR和SAR反应，增强了菜豆对FOP-DM01菌株的抗性。更多还原

【Abstract】 Common bean is one of the main legumes for human consumption with high nutritional value andlarge-scale planting area. However, Fusarium wilt （FW） was a serious disease in the agricultrualproduction of common bean and usually brought greater losses to common bean production in thedisease happened year. Therefore, it was significant to ensure the food security and improve theeconomic value of common bean production by screening for stable resistance germplasm resources ofcommon bean, revealing resistance mechanism of the common beans to FW, discovering and takingadvantage of excellent resistance gene so as to improve the resistance of different common beanvarieties. In this study, a transcription factor gene （ftf1genes） from Fusarium oxysporum f. sp. phaseoliwhich is closely related to its virulence was used to design a pair of specific primers. The primers werecombined with real time quantitative PCR to differentiate the resistance level of different common beancultivars accurately. It revealed differentially expressed genes in interaction between common bean andpathogen by cDNA-AFLP analysis, then we cloned two defence-response genes named PvCaM1encoding calmodulin and PvPOX1encoding secretory peroxidase, separately. The exogenous planthormone was used to induce the host resistance and reveal the resistance signalling pathway andresistance mechanism of the common bean to FW so as to supply the gene resources and theoreticalbasis for resistance genetic improvement. The results as followed:1.59Fusarium oxysporum isolates collected from8locations in6provinces were identifiedusing pathogenicity test, morphological and molecular biological methods. The results indicated59F.oxysporum contained37pathogenic isolates. A transcription factor gene （ftf1genes） from Fusariumoxysporum f. sp. phaseoli which is closely related to its virulence was used to design a pair of specificprimers QFopA/QFopB, and149bp amplified product was obtained only from F. oxysporum f. sp.phaseoli. Therefore, the primers was used to identify F. oxysporum f. sp. phaseoli and screening for theresistant resource quantitatively.2.362accessions of common bean germplasm resources were screened for the resistant resourceinoculated with FOP-DM01isolate by the corn meal inoculum after comparation with the hypocotylsdouble holes injection and the root dipped inoculum, and we obtained16resistant cultivars, and2ofthem named with260205and Heiyundou showed stable resistance level, their accession numbers wereF0005035and F0004851, respectively. Using the specific primers QFopA/QFopB, the minimum of1pgpathogen DNA was detected by real time-PCR. The result indicated that FOP-DM01DNAquantifications in the roots and stems of susceptible BRB-130and A0640-1were significantly higherthan those in resistant260205and Heiyundou, which absolutely matched with the phenotypicidentification.3. The differentially expressed genes in interaction between common bean and pathogen wereanalyzed by cDNA-AFLP. Totally443differentially expressed fragments were obtained, and172ofthem were sequenced. The result indicated that a large number of plant resistance-related genes wasobtained, such as the GH3auxin-regulated protein, calmodulin, peroxidase and the expression level wasdeterminated by real time quantitative PCR; The results of scanning electron micrographs （SEM） and transmission electron micrographs （TEM） showed that the pathogens caused serious damage to theorganizational and celluar structure of roots and stems in BRB-130, however, the260205root and steminfected by pathogen were not obviously damaged; Phenylalanine ammonia lyase （PAL） and peroxidase（POX） activity was determinated, the result indicated that the activities of the two enzymes in the rootand stem of260205were significantly higher than BRB-130, which showed that these two plantdefense-related enzymes played an important role in the resistance of common bean to FW. In addition,H₂O₂and O₂^-content in the root and stem of260205and BRB-130were measured, it showed H₂O₂andO₂^-of260205were higher than BRB-130both in root and stem. Therefore, reactive oxygen species（ROS） were testified as an important signalling molecular in the resistant response of common bean.4. A full-length cDNA sequence coding for CaM in common bean was cloned based onexpressed sequence tags from common bean, designated PvCaM1（GenBank accession numberJN418801）. Phylogenetic analysis based on the amino acids sequence of PvCaM1showed that theprotein encoded by this gene had the closest relationship with the CaM in Lotus japonicus andwatermelon, the homology were77%and76%, respectively. Real time-PCR analysis indicated that theexpression level of PvCaM1in the interactions between common bean and FOP-DM01isolateincreased significantly. The expression level of PvCaM1in leaves was higher than in roots.Transcriptional level of PvCaM1was up-regulated by exogenous abscisic acid（ABA）, methyljasmonate（MeJA） and ethephon（ETH）. PvCaM1expressed differentially in the leaves, stems and roots,and the expression level in leaves was higher than that in roots and stems.5. A full-length cDNA sequence coding for POX in common bean was cloned based onexpressed sequence tags from common bean, designated PvPOX1（GenBank accession numberJQ627838）. Phylogenetic analysis based on the amino acids sequence of PvPOX1showed that theprotein encoded by this gene had the closest relationship with the POX in soybean and purple medic, thehomology were90%and85%, respectively. Real time-PCR analysis indicated that the enhancedexpression level of PvPOX1significantly correlated with the change of POX activity and H₂O₂content.PvPOX1expressed differentially in the roots, stems, leaves, flowers and pods, the expression level ofleaves was the lowest, and that of pods was the highest. Transcriptional level of PvPOX1wasup-regulated by exogenous salicylic acid（SA）, ABA and other abiotic stress such as mechanicalwounding, salt and drought.6. SA, MeJA, ETH, paclobutrazol and nordihydroguaiaretic acid（NDGA） were used to treatBRB-130plants. The results indicated that plants treated by SA on leaves induced SA level in rootsincreased, and significantly enhanced the plant resistance to FOP-DM01isolate. In addition, SAinhibited growth of the pathogen in roots by inducing SAR in plants significantly, and SA had beentested to have no direct antifungal activity to FOP-DM01isolate in vitro; SA enhanced the activities ofPAL and POX, and increased H₂O₂and O₂^-content in common bean root significantly. These enhancedthe resistance to FOP-DM01isolate through induction of HR and SAR of common bean.更多还原