【作者】 刘东；

【导师】 秦智伟；

【作者基本信息】 东北农业大学 ， 蔬菜学， 2017， 博士

【摘要】 黄瓜（Cucumis sativus L.）是一种以嫩果为食用的大宗蔬菜。黄瓜属于草本科蔓生植物,在生产中易受多种病原菌的侵染,使其产量和品质受到严重影响。黄瓜霜霉病和棒孢叶斑病是黄瓜生产上的重要病害。病害传播流行速度快,对黄瓜生产威胁极大。长期使用化学药剂进行病害防治,已经导致霜霉病菌和棒孢叶斑病菌产生抗药性。同时大量的农药残留会造成环境污染和危害人类健康等问题。绿色、经济有效的防治方法仍为选育及利用抗病品种。培育单一抗病品种已无法解决病害给黄瓜生产带来的严重影响。国内外对黄瓜棒孢叶斑病的研究基础比较薄弱,多集中在病原菌生理分化、抗药性及抗性遗传规律等方面,缺少系统的抗病机理方面的研究。黄瓜霜霉病的研究起步虽早,但抗性遗传规律仍存在争议,分子调控机理尚不明了。本研究从抗源筛选、组织病理学、转录组学和基因功能方面对黄瓜双抗机制进行了研究,主要结果如下:（1）采用苗期人工接种鉴定法,对50份黄瓜种质资源进行霜霉病和棒孢叶斑病抗病性鉴定。结果表明,无高抗霜霉病材料,抗霜霉病材料有11份;高抗棒孢叶斑病材料有9份。通过对50份材料双抗性比较,抗2种病害材料共有5份,品种编号分别为16号（欧洲鲜用型）、28号（腌渍型）、31号（华北型）、38号（华南型）和50号（华南型）,占总数的10%。在4种生态类型中,华南型黄瓜双抗性最好;筛选获得双抗材料（38号）和双感材料（6号）用于后续研究。（2）利用光学显微镜观察霜霉病菌侵染抗感黄瓜品种的侵染过程,结果表明霜霉病菌通过游动孢子萌发产生芽管进行侵染,侵染进程在抗感黄瓜品种上没有差异;接种24 h芽管可从气孔侵入黄瓜叶片组织。采用离体接种和台盼蓝染色观察HR发生情况,结果表明抗病品种D9320接种36 h,显微镜下观察到HR现象,接种4 d产生肉眼可见坏死斑;感病品种D0401接种5 d,显微镜下观察到微弱的HR现象,但并无肉眼可见坏死斑产生;接种5 d,抗病品种D9320上菌丝蔓延量明显少于感病品种D0401。HR的发生对霜霉病菌的侵染起到了抑制作用。（3）利用光学显微镜及扫描电镜对棒孢叶斑病菌侵染抗感黄瓜品种过程进行观察,结果表明侵染进程在抗感品种中没有差别。棒孢叶斑病菌通过分生孢子萌发产生芽管进行侵染,24 h芽管可直接侵入或从气孔侵入黄瓜叶片组织。采用离体接种和台盼蓝染色观察HR,结果显示抗病品种D9320接种24 h,显微镜下观察到HR现象,接种4 d产生肉眼可见坏死斑;感病品种D0401接种5 d,显微镜下观察到微弱的HR现象,但并无肉眼可见坏死斑产生。接种5 d,抗病品种D9320上菌丝蔓延量明显少于感病品种D0401。HR的发生对棒孢叶斑病菌的侵染起到了抑制作用。（4）采用DAB、TB和AB染色,显微观察H₂O₂、木质素和胼胝质的积累。观察结果显示,接种霜霉病菌和棒孢叶斑病菌后三者的积累模式一致,抗病品种D9320中的积累均早且高于感病品种D0401;不同点在于抗病品种D9320接种棒孢叶斑病菌后三者的积累稍早于接种霜霉病菌后的积累。（5）利用转录组测序（RNA-seq）技术和比较基因组学的方法对不同病原菌胁迫下的差异表达基因进行分析,共得到45个在抗病品种中上调,感病品种中下调或非差异的基因。根据基因注释结果,筛选到与植物抗病性关系密切的10个基因,分别为果胶裂解酶基因、蛋白激酶家族基因、BAG家族基因、CBS结构域蛋白基因、赤霉素调节蛋白基因、ERF转录因子、b HLH157转录因子、锌指蛋白基因、钙调素蛋白基因和CCT基序蛋白基因。其中ERF转录因子在植物抗病反应的转录调控中具有重要作用,筛选进行功能分析。（6）在不同病原菌胁迫下,对抗病品种D9320和感病品种D0401中差异表达基因进行pathway显著性富集分析,结果显示抗感品种中共有的代谢通路为19个。差异基因所占比例较高且与植物抗病性关系密切的pathway有植物病原菌互作（Plant-pathogen interaction）苯丙氨酸代谢（Phenylalanine metabolism）、类黄酮生物合成（Flavonoid biosynthesis）和次生代谢产物的生物合成（Biosynthesis of secondary metabolites）。（7）利用PCR技术克隆了黄瓜Cs ERF004基因,并对其进行生物信息学分析。结果表明Cs ERF004基因编码蛋白具有1个AP2结构域,属于ERF亚组中B-6类蛋白;启动子序列预测分析发现,具有防御及应激反应元件、真菌激发子响应元件、乙烯应答元件和水杨酸响应元件等多个反应调控元件,推测黄瓜Cs ERF004基因表达可能与黄瓜抗病性相关。（8）利用qRT-PCR技术分析黄瓜Cs ERF004基因在不同病原菌胁迫下的表达模式。结果显示接种棒孢叶斑病菌、霜霉病菌及同时接种2种病原菌的3种处理下,Cs ERF004在抗病品种D9320中呈现特异表达模式,即在病原菌胁迫下从2 h到96 h呈显著上调表达,96 h到达高峰之后开始下降,整体呈抛物线变化趋势;而在感病品种D0401中,其表达量很低且几乎无变化,仅相当于D9320中2 h的表达水平,从2 h到120 h的表达量呈直线形。结果显示Cs ERF004积极响应霜霉病菌和棒孢叶斑病菌的胁迫,可能对2种病原菌的侵染具有一定抵抗能力,并参与黄瓜双抗性的抗病反应。（9）利用qRT-PCR技术分析黄瓜Cs ERF004基因在不同激素诱导下的表达模式。结果显示黄瓜Cs ERF004基因能够在水杨酸和乙烯的诱导下上调表达,不能被茉莉酸诱导表达。（10）成功构建Cs ERF004基因与绿色荧光蛋白（GFP）的融合表达载体,通过绿色荧光蛋白（GFP）瞬时表达系统分析检测,Cs ERF004在细胞核中表达。（11）成功构建Cs ERF004基因过表达载体。利用农杆菌介导法将构建的Cs ERF004基因过量表达载体导入黄瓜感病品种D0401中,获得转基因植株。经霜霉病菌和棒孢叶斑病菌接种鉴定,结果表明过表达Cs ERF004的T0代和T1代植株都能显著提高黄瓜对霜霉病及棒孢叶斑病的抗性,T1代植株病情指数下降比率均达到30%以上,同时防御基因Cs PR1和Cs PR4显著上调表达且SA及ET含量增加。更多还原

【Abstract】 Cucumber（Cucumis sativus L.）is a staple vegetable that produces tender edible fruits.In production,cucumber is susceptible to a variety of infections,thus severely affecting its yield and quality.Two major diseases affecting cucumber production include cucumber downy mildew and cucumber target spot.Disease epidemics involving these pathogens generally spread fast,thereby disrupting cucumber production.Corynespora cassiicola and Pseudoperonospora cubensis isolates from cucumber that are resistant to fungicides have arisen because many fungicides have been used for a long time.A large number of pesticide residues will pose a serious threat to the environment and human health.The breeding and utilization of resistant varieties is still the safest and most efficient way in controlling diseases.The use of single cucumber disease-resistant varieties has been determined to be insufficient in fully compensating for the losses in cucumber production due to these infectious diseases.Research on cucumber target spot is relative weak,which has focused on the physiological specialization of the pathogen,resistance to chemical fungicides and resistance genetic regulation,among other topics,and lacked the system study on the mechanism of disease resistance.Although the study of cucumber downy mildew started early,resistance genetic regulation is still controversial and the mechanism of molecular regulation is unclear.In this study,we investigated the double-disease resistance mechanism of cucumber from the aspects of resistance screening,histopathology,transcriptome and gene function,The main results are as follows,（1）Seedling-inoculation method was used to identify the resistance of 50 germplasms to downy mildew and target spot.The results showed that there were 11 resistant materials to downy mildew and 9 high resistant materials to target spot.Analysis of double diseases resistance suggested that 5 materials were resistant to double diseases.They were No.16（Eurpean fresh type）,No.28（pickling type）,No.31（North China type）,No.38（South China type）and No.50（South China type）,10 percent of the total.The double diseases resistance of South China type cucumber was the best in four ecological types.（2）The process of P.cubensis infection was observed using a microscope.The results showed that there were no differences in infection process between these cultivars.The germ tube invaded the cucumber leaf tissue via stomata at 24 hai.The hypersensitive response（HR）was observed using in vitro inoculation and the trypan blue staining method.HR was observed at 36 hai in D9320 in the light microscope,and necrotic lesions occurred at 4 dpi in D9320 in vitro inoculation.HR was very weak at 5 dpi in D0401 in the light microscope,and necrotic lesions did not occur in D0401 in vitro inoculation.The amount of mycelium in D9320 was much less than that in D0401 at 5 dpi.The phenomena revealed that stronger HR can limit the growth of P.cubensis.（3）The process of C.cassiicola infection was observed using a microscope and a scanning-electron microscope.The results showed that there were no differences in infection process between these cultivars.The germ tube rapidly elongated within 24 hours after inoculation and invaded the cucumber leaf tissue directly or via stomata 24 hours after inoculation（hai）.The hypersensitive response（HR）was observed using in vitro inoculation and the trypan blue staining method.HR was observed at 24 hai in D9320 in the light microscope,and necrotic lesions occurred at 4 dpi in D9320 in vitro inoculation.HR was very weak at 5 dpi in D0401 in the light microscope,and necrotic lesions did not occur in D0401 in vitro inoculation.The amount of mycelium in D9320 was much less than that in D0401 at 5 dpi.The phenomena revealed that stronger HR can limit the growth of C.cassiicola.（4）The accumulation of H₂O₂,lignin and callose was observed using DAB,TB and AB staining.The results showed that the pattern of H₂O₂,lignin and callose accumulation were the same in D9320 and D0401 infected with C.cassiicola and P.cubensis.H₂O₂,lignin and callose accumulation in D9320 occurred earlier and stronger than in D0401.The difference was that their accumulations were earlier in D9320 infected with C.cassiicola than in D9320 infected with P.cubensis.（5）Using RNA-seq technology,differentially expressed genes under different pathogens stress were analysed through the method of comparative genomics.45 genes were upregulated in the resistant cultivar D9320,and downregulated or non-differentiated in the susceptible cultivar D0401.10 genes were determined to be related to disease resistance by gene annotation,which were pectin lyase gene,protein kinase family gene,BAG family gene,CBS domain protein gene,gibberellin regulated protein gene,ERF transcription factor,b HLH157 transcription factor,zinc finger protein gene,calmodulin gene and CCT motif protein gene.（6）Under different pathogens stress,the significant enrichment pathway of differentially expressed genes was analysed.The resulted showed that 19 pathways were obtained in D9320 and D0401.Pathways related to disease resistance were plant-pathogen interaction,phenylalanine metabolism,flavonoid biosynthesis and biosynthesis of secondary metabolites.（7）The Cs ERF004 gene was cloned by PCR,and its bioinformatics analysis was carried out.The results showed that the protein encoded by Cs ERF004 gene has one AP2 domain,and belongs to the ERF transcription factor B-6 family.The results of analysis of promoter indicated that it had cis-elements involved in defense and stress response element,fungal elicitor response element,ethylene responsive element and salicylic acid responsive element.It is speculated that the expression of Cs ERF004 gene may be related to the disease resistance of cucumber.（8）Using the q RT-PCR technology,the expression of Cs ERF004 was determined under pathgen stress.The three treatments,namely,a single inoculation of C.cassiicola,P.cubensis,and simultaneous inoculation of two pathogens,resulted in a pecific expression pattern of Cs ERF004 in the resistant cultivar D9320 that was such as a parabola.The expression level increased from 2 h to 96 h,and reached a peak at 96 h,and then decreased slightly.In the susceptible cultivar D0401,the expression pattern was such as a horizontal line.The expression level had almost unchanged.Cs ERF004 gene had the most positive response to stress on infection of C.cassiicola and P.cubensis.Cs ERF004 gene might be closely related to resistance to downy mildew and target spot.（9）Using the q RT-PCR technology,the expression of Cs ERF004 was determined under hormone induction.The results showed that Cs ERF004 could be upregulated by SA and ET,but it did not significantly change in Me JA induction.（10）The Cs ERF004 with GFP-fusion protein expression vector was successfully constructed,Cs ERF004 localized in the nucleu by transient expressing of GFP-fusion protein.（11）Overexpression vectors of Cs ERF004 was successfully constructed.The overexpression vector p CXSN-Cs ERF004 was transferred into the susceptible cultivar D0401.The result of resistance identification showed that over-expression of Cs ERF004 in T0 and T1 lines could improve the resistance of cucumber to downy mildew and target spot,and the decreasing ratio of disease index of T1 generation plants was more than 30%.Further analysis indicated that overexpression of Cs ERF004 in susceptible cultivar D0401 resulted in a significant upregulation of the Cs PR1 and Cs PR4 genes and increased levels of SA and ET.更多还原