银杏内生细菌防治辣椒疫病机制研究
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摘要
本研究从山东、河南、浙江及福建四个地区采集不同生长年龄的健康银杏叶片进行内生细菌的分离，共获得120株内生细菌，来自山东泰安约50年生银杏植株获得21株，来自河南郑州约30年生获得62株，来自福建福州约10年生获得17株，来自浙江杭州约100年生获得20株。分离结果表明银杏叶片内存在大量的内生细菌，且不同树龄银杏叶片内生细菌的种群密度和含量存在显著差异，表现为约50年生＞约30年生＞约10年生＞约100年生。利用16S rDNA PCR-RFLP、ERIC-PCR和DNA gyrase B subunit（gyrB）基因测序的方法分析银杏内生细菌的种群多样性，结果表明银杏叶片内生细菌具有丰富的种群多样性。16S rDNAPCR-RFLP分析结果表明120株内生细菌菌株共产生42种酶切图谱类型，具有优势RFLP图谱类型的64个菌株有53个ERIC-PCR图谱类型，表明银杏优势内生细菌具有相对丰富的遗传型。将每一个RFLP图谱类型中的代表菌株进行gyrB基因测序，测序结果表明代表菌株共属于9个属26个种，其中芽胞杆菌属（Bacillus）和假单胞菌属（Pseudomonas）细菌为银杏叶片中内生细菌的优势种群。
通过平板对峙试验从来自银杏叶片的120株内生细菌中筛选获得35株对辣椒疫霉菌（Phytophthora capsici）具有拮抗作用的菌株，其中8个菌株Zy44、Fy11、Hy7、Hy14、Zy25、Zy45、Zy55和Sy15对辣椒疫霉菌拮抗活性较强，抑菌带的宽度均在10mm以上。进一步测定8个内生细菌菌株对其他一些供试植物病原菌的平板拮抗作用，结果表明这8个菌株对测试的病原菌均有不同程度的抑制作用。继续测定8个拮抗菌株对辣椒果疫病的防治效果，发现8个测试菌株对辣椒果疫病均有不同程度的防病作用，其中5个菌株Zy44、Fy11、Hy7、Hy14和Zy25防效显著，挑战接种病原菌6d后，防效仍保持在70%以上。温室盆栽辣椒苗疫病的防治试验表明，菌株Zy44和Fy11对辣椒苗疫病具有较好的防治效果，接种病原菌20d后，两个菌株的防效保持在50%以上。经形态观察、生理生化特征测试、16S rDNA序列和gyrB基因序列分析，鉴定4个生防菌株Zy44、Fy11、Hy7和Zy25为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。
三个生防菌株Fy11、Hy7和Zy44混配后对辣椒果疫病和苗疫病的防治试验表明，Fy11和Zy44混配后能够显著增加辣椒果疫病和苗疫病的防治效果，接种病原菌7d后，对辣椒果疫病的防病效果仍保持在76.30%，接种病原菌20d后，对辣椒苗疫病的防病效果为71.30%。温室盆栽辣椒苗的促生试验表明，3个生防菌株均能显著促进辣椒幼苗的生长，菌株Fy11、Hy7和Zy44对辣椒苗的促生增长率分别为43.65%、39.53%、46.93%。应用绿色荧光蛋白GFP基因标记生防菌株，工程菌株Fy11-gfp和Zy44-gfp在辣椒组织内的定殖研究表明，两个生防菌株均可在辣椒组织内定殖，能够从根部运输到茎和叶。在整个分离过程中，菌株Fy11的定殖量显著高于菌株Zy44，尤其是辣椒的茎和叶组织中。同时研究发现Fy11和Zy44混合后接种辣椒幼苗后，其定殖数量与单个生防菌株的定殖数量并没有显著差异。进一步对菌株Fy11和Zy44胞外代谢物质的防病效果进行研究，结果发现菌株Zy44的发酵滤液和脂肽类粗提物对辣椒苗疫病的防治效果显著高于菌株Fy11的发酵滤液和脂肽类粗提物，同时Zy44脂肽类粗提物对辣椒苗疫病的防治效果高于该菌株的发酵滤液。光学显微镜下观察发现Zy44的脂肽类粗提物能够导致辣椒疫霉菌菌丝畸形，并抑制其游动孢子囊的形成。在研究生防菌株Fy11和Zy44诱导辣椒系统抗性时，荧光定量PCR的结果表明内生细菌浇灌处理，再挑战接种病原菌后，菌株Fy11能够诱导辣椒防病相关基因的表达，显著增强防病基因CaPR4（辣椒病程相关蛋白4）的表达，Zy44处理、SA处理以及病原菌处理后辣椒幼苗防病相关基因的表达量基本相同。
利用合成扩增枯草芽胞杆菌功能基因bmyB、fenD、ituC、srfAA、srfAB和bioA、yngG和yndJ的8对特异引物PCR扩增拮抗芽胞杆菌Zy25和Hy7的生防功能基因，发现均可以扩增获得8个生防基因片段，表明菌株Zy25和Hy7可能具有合成脂肽类物质的能力。平板拮抗作用测试表明其合成的脂肽类物质可抑制多种植物病原真菌的生长，光学显微镜下观察发现拮抗芽胞杆菌的脂肽类粗提物能够导致植物病原真菌菌丝畸形，原生质凝结，无法扩展生长。活体生防试验表明Zy25和Hy7脂肽类粗提物能够有效的防治辣椒果疫病和番茄果实灰霉病。
利用cDNA-AFLP技术，采用256对引物，分析辣椒幼苗接种内生解淀粉芽胞杆菌Fy11后5个时间点的基因表达谱，初步明确内生解淀粉芽胞杆菌Fy11诱导辣椒幼苗差异表达基因。结果表明256对引物共产生18620个转录本（TDFs），筛选得到353条差异表达条带，占扩增条带总数的1.89%。经克隆、测序分析，最终获得257个差异TDFs，聚类分析得到229个ESTs（unigenes），其中144个基因上调表达，85个基因下调表达。经Blastx比对和功能分类分析，其中65条EST（s28.38%）未找到同源性匹配，8条（3.49%）与未知功能蛋白同源性较高。其余156条ESTs主要涉及基础代谢基因（10.92%）；与能量和抗病与防御类相关基因，各占8.73%；信号转导基因占7.42%；转运子或转座子基因占6.99%；与细胞结构相关基因占6.55%；参与转录调控基因占5.68%；细胞生长类基因占3.93%；参与蛋白质运输和储存8个基因，占3.49%；蛋白质合成类占2.18%；次生代谢类和胞间运输类基因，其数量相对较少，各占1.75%。选取抗病与防御、转录调控及信号转导类等相关的10个差异基因，利用qRT-PCR分析差异基因表达模式，验证cDNA-AFLP表达谱，qRT-PCR分析结果显示其表达模式符合cDNA-AFLP表达谱。
The120endopytic bacterial isolates were isolated from surface-sterilized leaves ofGinkgo biloba, which collected from Hangzhou, Zhejiang province, Zhengzhou, Henanprovince, Taian, Shandong province and Fuzhou Fujian province. The21bacterialisolates isolated from about50-year-old Ginkgo biloba leaves(Shandong province),62bacterial isolates isolated from about30-year-old Ginkgo biloba leaves(Henan province),17bacterial isolates isolated from about10-year-old Ginkgo biloba leaves(Fujianprovince),20bacterial isolates isolated from about100-year-old Ginkgo bilobaleaves(Zhejiang province).The results indicated that many endopytic bacteria existed inGinkgo biloba leaves and the endopytic bacterial populations had a significant differencein Ginkgo biloba leaves of different tree ages, that is about50-year-old＞about30-year-old＞about10-year-old＞about100-year-old. Using16S rDNA PCR-RFLP、ERIC-PCR and DNA gyrase B subunit （gyrB） sequence analysis, genetic diversity ofthe endophytic bacteria within Ginkgo biloba leaves were explored. The results showedthat the endopytic bacteria had a rich population diversity in Ginkgo biloba leaves.The120strains obtained42restriction endonuclease types based on the16S rDNAPCR-RFLP analysis.Further, the64isolates having predominant genotype were dividedinto53ERIC-PCR fingerprintings.The results showed the genetic diversity ofpredominant endophytic strains was high. The gyrB sequences of representative strains ofeach RFLP types were tested and the phylogenic trees were constructed.The resultsrevealed that these isolates belonged to26species of9different genera, and endopyticbacterial isolates belonged to genera of Pseudomonas and Bacillus that were thepredominant genera.
Based on screening in vitro, the35bacterial isolates among120strains showedobvious inhibition ability on the mycelia of Phytophthora capsici.Especially, the strainsZy44、Fy11、Hy7、Hy14、Zy25、Zy45、Zy55and Sy15showed strong inhibitory activitywith more than10mm inhibition zones on plates. The8isolates showed differentinhibiting effect on plant pathogens in the dual plate assays. Further, the8isolates werechosen to control fruit phytophthora blight of pepper. The results showed that all thetested isolates could control the disease with different extent, and the isolates, Zy44、Fy11、Hy7、Hy14and Zy25showed significant biocontrol activity to the disease withmore than70%control effect in6d after pathogen challenge. The5isolates wereevaluated their biocontrol effects on seedling phytophthora blight of pepper ingreenhouse experiments. The isolates, Zy44and Fy11, were found to have betterbiocontrol efficacy with more than50%control effect in20d after pathogen challenge. Based on the morphological, physiological, biochemical characteristics,16S rDNA andgyrB sequence analysis, the strains Zy44、Fy11、Hy7and Zy25were identified as Bacillusamyloliquefaciens.
The3biocontrol strains, Fy11、Hy7and Zy44, were tested in combinations forbiological control against fruit and seedling phytophthora blight of pepper. The mixturesof Fy11and Zy44showed significant reductions in disease severity on fruits andseedlings phytophthora blight of pepper after pathogen challenge, comparing with othercombinations and a single strain The control effects were76.3%and71.3%respectively,.Further, the3strains were evaluated their growth promotion effects on pepper seedling ingreenhouse experiments. The results indicated that the endophytic strains, Fy11、Hy7andZy44, were also effective for promoted the growth of pepper seedlings with43.65%、39.53%and46.93%growth-promoting efficacy after treated in30days,respectively. Thebiocontrol strains, Fy11and Zy44，were labeled with green fluorescent protein gene toinvestigate the colonization of the bacteria in the pepper. The2strains were able tocolonize in the internal tissues of the pepper and transport into stem and leave from root.The colonization density of Fy11was higher than that of Zy44in the pepper tissuethroughout the duration of the experiment, especially stem and leaves of the pepper. Theendophytic colonization of mixtures of two strains was not significant difference incontrast to a single strain. Direct antagonism of antifungal compounds produced byendophytic bacteria was investigated.The cell-free filtrate and lipopetides produced bythe strain Zy44was able to suppress effectively seedling phytophthora blight of pepper,and the lipopetides compounds were better than cell-free filtrate, but direct antagonism ofculture filtrate and lipopetides produced by Fy11was not obvious. It was observed bylight microscopy that the lipopetides compounds produced by the strains Zy44couldinduce alternations on hyphae morphology and reducing the sporangia production ofP.capsici. The induced systemic resistance (ISR) mediated by Fy11and Zy44on pepperwas further analysed using priming effect of the defense-related genes after pathogenchallenge. qRT-PCR analyses revealed that the expression of the defense-related geneswas primed by the application of strain Fy11, especially, the expression of the CaPR4(Capsicum annuum pathogenesis-protein4) gene, but the expression levels ofdefense-related genes were not obvious different in plants treated with Zy44-, SA-, andthe pathogen.
The biocontrol function gene fragments of bmyB, fenD, bioA, srfAA, srfAB, bioA,yngG and yndJ were amplified with specific primers from genomic DNA of the twobiocontrol strains, Zy25and Hy7, which indicated that the two strains could producelipopetide metabolites. The lipopetides compounds produced by Zy25and Hy7exhibitedinhibitory activity on mycelium growth of some plant pathogens detected. The mycelia of plant pathongens treated with lipopetides compounds appeared distortion，protoplasmagglomerated and stopped growing，which were observed by light microscopy. Thelipopetides compounds produced by Zy25and Hy7were evaluated their biocontroleffects on fruit of pepper phytophthora blight and tomato gray mould.The results showedthat lipopetides compounds produced by Zy25and Hy7were able to reduce effectivelydisease sevisity.
cDNA-AFLP technique was used to conduct transcription profiling of pepperseedlings across five sampling time points after inoculation with the strain Fy11. Theresult showed that the total18620transcript derived fragments (TDFs) were obtained byamplification with256primer pairs, among which353(1.89%) displayed alteredexpression patterns after inoculation. Two hundred and fifty seven differentiallyexpressed TDFs produced reliable sequences after cloning and sequencing. Two hundredand twenty nine expressed sequence tags (ESTs) of unigenes were obtained afterassembling, of which144showed up-regulated and85down-regulated. Blastx analysesand functional annotations were then performed and the results revealed that the156ESTs had predicted gene products mainly implicated in energy (10.92%), metabolism anddisease/defense (each accounted for8.73%), signal transduction (7.42%),transporter(6.99%), cell structure(6.55%), transcription(5.68%), cell growth (3.93%),protein destination and storage(3.49%), protein synthesis (2.18%), secondary metabolismand intracellur traffic (each accounted for1.75%). The65EST（s28.38%of the sequencedtotal229ESTs) had no match to known genes, the8ESTs(3.49%of the sequenced total229ESTs) were highly homologous to unknown functional proteins. Ten differentialgenes related to disease/defense, transcription and signal transduction were chosen forfurther qRT-PCR expression patterns to evaluate the validation of cDNA-AFLP. Theresults showed that these genes expression patterns confirmed the cDNA-AFLPprofiles.
通过平板对峙试验从来自银杏叶片的120株内生细菌中筛选获得35株对辣椒疫霉菌（Phytophthora capsici）具有拮抗作用的菌株，其中8个菌株Zy44、Fy11、Hy7、Hy14、Zy25、Zy45、Zy55和Sy15对辣椒疫霉菌拮抗活性较强，抑菌带的宽度均在10mm以上。进一步测定8个内生细菌菌株对其他一些供试植物病原菌的平板拮抗作用，结果表明这8个菌株对测试的病原菌均有不同程度的抑制作用。继续测定8个拮抗菌株对辣椒果疫病的防治效果，发现8个测试菌株对辣椒果疫病均有不同程度的防病作用，其中5个菌株Zy44、Fy11、Hy7、Hy14和Zy25防效显著，挑战接种病原菌6d后，防效仍保持在70%以上。温室盆栽辣椒苗疫病的防治试验表明，菌株Zy44和Fy11对辣椒苗疫病具有较好的防治效果，接种病原菌20d后，两个菌株的防效保持在50%以上。经形态观察、生理生化特征测试、16S rDNA序列和gyrB基因序列分析，鉴定4个生防菌株Zy44、Fy11、Hy7和Zy25为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。
三个生防菌株Fy11、Hy7和Zy44混配后对辣椒果疫病和苗疫病的防治试验表明，Fy11和Zy44混配后能够显著增加辣椒果疫病和苗疫病的防治效果，接种病原菌7d后，对辣椒果疫病的防病效果仍保持在76.30%，接种病原菌20d后，对辣椒苗疫病的防病效果为71.30%。温室盆栽辣椒苗的促生试验表明，3个生防菌株均能显著促进辣椒幼苗的生长，菌株Fy11、Hy7和Zy44对辣椒苗的促生增长率分别为43.65%、39.53%、46.93%。应用绿色荧光蛋白GFP基因标记生防菌株，工程菌株Fy11-gfp和Zy44-gfp在辣椒组织内的定殖研究表明，两个生防菌株均可在辣椒组织内定殖，能够从根部运输到茎和叶。在整个分离过程中，菌株Fy11的定殖量显著高于菌株Zy44，尤其是辣椒的茎和叶组织中。同时研究发现Fy11和Zy44混合后接种辣椒幼苗后，其定殖数量与单个生防菌株的定殖数量并没有显著差异。进一步对菌株Fy11和Zy44胞外代谢物质的防病效果进行研究，结果发现菌株Zy44的发酵滤液和脂肽类粗提物对辣椒苗疫病的防治效果显著高于菌株Fy11的发酵滤液和脂肽类粗提物，同时Zy44脂肽类粗提物对辣椒苗疫病的防治效果高于该菌株的发酵滤液。光学显微镜下观察发现Zy44的脂肽类粗提物能够导致辣椒疫霉菌菌丝畸形，并抑制其游动孢子囊的形成。在研究生防菌株Fy11和Zy44诱导辣椒系统抗性时，荧光定量PCR的结果表明内生细菌浇灌处理，再挑战接种病原菌后，菌株Fy11能够诱导辣椒防病相关基因的表达，显著增强防病基因CaPR4（辣椒病程相关蛋白4）的表达，Zy44处理、SA处理以及病原菌处理后辣椒幼苗防病相关基因的表达量基本相同。
利用合成扩增枯草芽胞杆菌功能基因bmyB、fenD、ituC、srfAA、srfAB和bioA、yngG和yndJ的8对特异引物PCR扩增拮抗芽胞杆菌Zy25和Hy7的生防功能基因，发现均可以扩增获得8个生防基因片段，表明菌株Zy25和Hy7可能具有合成脂肽类物质的能力。平板拮抗作用测试表明其合成的脂肽类物质可抑制多种植物病原真菌的生长，光学显微镜下观察发现拮抗芽胞杆菌的脂肽类粗提物能够导致植物病原真菌菌丝畸形，原生质凝结，无法扩展生长。活体生防试验表明Zy25和Hy7脂肽类粗提物能够有效的防治辣椒果疫病和番茄果实灰霉病。
利用cDNA-AFLP技术，采用256对引物，分析辣椒幼苗接种内生解淀粉芽胞杆菌Fy11后5个时间点的基因表达谱，初步明确内生解淀粉芽胞杆菌Fy11诱导辣椒幼苗差异表达基因。结果表明256对引物共产生18620个转录本（TDFs），筛选得到353条差异表达条带，占扩增条带总数的1.89%。经克隆、测序分析，最终获得257个差异TDFs，聚类分析得到229个ESTs（unigenes），其中144个基因上调表达，85个基因下调表达。经Blastx比对和功能分类分析，其中65条EST（s28.38%）未找到同源性匹配，8条（3.49%）与未知功能蛋白同源性较高。其余156条ESTs主要涉及基础代谢基因（10.92%）；与能量和抗病与防御类相关基因，各占8.73%；信号转导基因占7.42%；转运子或转座子基因占6.99%；与细胞结构相关基因占6.55%；参与转录调控基因占5.68%；细胞生长类基因占3.93%；参与蛋白质运输和储存8个基因，占3.49%；蛋白质合成类占2.18%；次生代谢类和胞间运输类基因，其数量相对较少，各占1.75%。选取抗病与防御、转录调控及信号转导类等相关的10个差异基因，利用qRT-PCR分析差异基因表达模式，验证cDNA-AFLP表达谱，qRT-PCR分析结果显示其表达模式符合cDNA-AFLP表达谱。
The120endopytic bacterial isolates were isolated from surface-sterilized leaves ofGinkgo biloba, which collected from Hangzhou, Zhejiang province, Zhengzhou, Henanprovince, Taian, Shandong province and Fuzhou Fujian province. The21bacterialisolates isolated from about50-year-old Ginkgo biloba leaves(Shandong province),62bacterial isolates isolated from about30-year-old Ginkgo biloba leaves(Henan province),17bacterial isolates isolated from about10-year-old Ginkgo biloba leaves(Fujianprovince),20bacterial isolates isolated from about100-year-old Ginkgo bilobaleaves(Zhejiang province).The results indicated that many endopytic bacteria existed inGinkgo biloba leaves and the endopytic bacterial populations had a significant differencein Ginkgo biloba leaves of different tree ages, that is about50-year-old＞about30-year-old＞about10-year-old＞about100-year-old. Using16S rDNA PCR-RFLP、ERIC-PCR and DNA gyrase B subunit （gyrB） sequence analysis, genetic diversity ofthe endophytic bacteria within Ginkgo biloba leaves were explored. The results showedthat the endopytic bacteria had a rich population diversity in Ginkgo biloba leaves.The120strains obtained42restriction endonuclease types based on the16S rDNAPCR-RFLP analysis.Further, the64isolates having predominant genotype were dividedinto53ERIC-PCR fingerprintings.The results showed the genetic diversity ofpredominant endophytic strains was high. The gyrB sequences of representative strains ofeach RFLP types were tested and the phylogenic trees were constructed.The resultsrevealed that these isolates belonged to26species of9different genera, and endopyticbacterial isolates belonged to genera of Pseudomonas and Bacillus that were thepredominant genera.
Based on screening in vitro, the35bacterial isolates among120strains showedobvious inhibition ability on the mycelia of Phytophthora capsici.Especially, the strainsZy44、Fy11、Hy7、Hy14、Zy25、Zy45、Zy55and Sy15showed strong inhibitory activitywith more than10mm inhibition zones on plates. The8isolates showed differentinhibiting effect on plant pathogens in the dual plate assays. Further, the8isolates werechosen to control fruit phytophthora blight of pepper. The results showed that all thetested isolates could control the disease with different extent, and the isolates, Zy44、Fy11、Hy7、Hy14and Zy25showed significant biocontrol activity to the disease withmore than70%control effect in6d after pathogen challenge. The5isolates wereevaluated their biocontrol effects on seedling phytophthora blight of pepper ingreenhouse experiments. The isolates, Zy44and Fy11, were found to have betterbiocontrol efficacy with more than50%control effect in20d after pathogen challenge. Based on the morphological, physiological, biochemical characteristics,16S rDNA andgyrB sequence analysis, the strains Zy44、Fy11、Hy7and Zy25were identified as Bacillusamyloliquefaciens.
The3biocontrol strains, Fy11、Hy7and Zy44, were tested in combinations forbiological control against fruit and seedling phytophthora blight of pepper. The mixturesof Fy11and Zy44showed significant reductions in disease severity on fruits andseedlings phytophthora blight of pepper after pathogen challenge, comparing with othercombinations and a single strain The control effects were76.3%and71.3%respectively,.Further, the3strains were evaluated their growth promotion effects on pepper seedling ingreenhouse experiments. The results indicated that the endophytic strains, Fy11、Hy7andZy44, were also effective for promoted the growth of pepper seedlings with43.65%、39.53%and46.93%growth-promoting efficacy after treated in30days,respectively. Thebiocontrol strains, Fy11and Zy44，were labeled with green fluorescent protein gene toinvestigate the colonization of the bacteria in the pepper. The2strains were able tocolonize in the internal tissues of the pepper and transport into stem and leave from root.The colonization density of Fy11was higher than that of Zy44in the pepper tissuethroughout the duration of the experiment, especially stem and leaves of the pepper. Theendophytic colonization of mixtures of two strains was not significant difference incontrast to a single strain. Direct antagonism of antifungal compounds produced byendophytic bacteria was investigated.The cell-free filtrate and lipopetides produced bythe strain Zy44was able to suppress effectively seedling phytophthora blight of pepper,and the lipopetides compounds were better than cell-free filtrate, but direct antagonism ofculture filtrate and lipopetides produced by Fy11was not obvious. It was observed bylight microscopy that the lipopetides compounds produced by the strains Zy44couldinduce alternations on hyphae morphology and reducing the sporangia production ofP.capsici. The induced systemic resistance (ISR) mediated by Fy11and Zy44on pepperwas further analysed using priming effect of the defense-related genes after pathogenchallenge. qRT-PCR analyses revealed that the expression of the defense-related geneswas primed by the application of strain Fy11, especially, the expression of the CaPR4(Capsicum annuum pathogenesis-protein4) gene, but the expression levels ofdefense-related genes were not obvious different in plants treated with Zy44-, SA-, andthe pathogen.
The biocontrol function gene fragments of bmyB, fenD, bioA, srfAA, srfAB, bioA,yngG and yndJ were amplified with specific primers from genomic DNA of the twobiocontrol strains, Zy25and Hy7, which indicated that the two strains could producelipopetide metabolites. The lipopetides compounds produced by Zy25and Hy7exhibitedinhibitory activity on mycelium growth of some plant pathogens detected. The mycelia of plant pathongens treated with lipopetides compounds appeared distortion，protoplasmagglomerated and stopped growing，which were observed by light microscopy. Thelipopetides compounds produced by Zy25and Hy7were evaluated their biocontroleffects on fruit of pepper phytophthora blight and tomato gray mould.The results showedthat lipopetides compounds produced by Zy25and Hy7were able to reduce effectivelydisease sevisity.
cDNA-AFLP technique was used to conduct transcription profiling of pepperseedlings across five sampling time points after inoculation with the strain Fy11. Theresult showed that the total18620transcript derived fragments (TDFs) were obtained byamplification with256primer pairs, among which353(1.89%) displayed alteredexpression patterns after inoculation. Two hundred and fifty seven differentiallyexpressed TDFs produced reliable sequences after cloning and sequencing. Two hundredand twenty nine expressed sequence tags (ESTs) of unigenes were obtained afterassembling, of which144showed up-regulated and85down-regulated. Blastx analysesand functional annotations were then performed and the results revealed that the156ESTs had predicted gene products mainly implicated in energy (10.92%), metabolism anddisease/defense (each accounted for8.73%), signal transduction (7.42%),transporter(6.99%), cell structure(6.55%), transcription(5.68%), cell growth (3.93%),protein destination and storage(3.49%), protein synthesis (2.18%), secondary metabolismand intracellur traffic (each accounted for1.75%). The65EST（s28.38%of the sequencedtotal229ESTs) had no match to known genes, the8ESTs(3.49%of the sequenced total229ESTs) were highly homologous to unknown functional proteins. Ten differentialgenes related to disease/defense, transcription and signal transduction were chosen forfurther qRT-PCR expression patterns to evaluate the validation of cDNA-AFLP. Theresults showed that these genes expression patterns confirmed the cDNA-AFLPprofiles.
引文
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