【作者】 刘龙洲；

【导师】 蔡润；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2008， 博士

【摘要】 黄瓜白粉病(powdery mildew,PM)是由专性活体寄生真菌引起,在温室和大田都普遍发生的叶部病害;其病原菌主要包括瓜单囊壳(Podosp -haera xanthii)和二孢白粉菌(Golovinomyces cichoracearum)两属;中国以瓜单囊壳危害较为普遍。白粉病病原菌寄主广泛,可以感染黄瓜植株叶片、茎和花,在开花期感染白粉病可减少产量30～50%。培育和应用抗性品种是抵御黄瓜白粉病危害的根本途径。据文献报道,黄瓜高抗白粉病品种较少,利用常规育种技术选育抗性品种有很大的困难。为了分子标记辅助育种和抗性基因分离,本文对黄瓜白粉病抗性进行了遗传分析和抗性基因定位研究。1.应用温室喷雾接种法,对从国内外收集的120份黄瓜种质材料进行两次重复接种鉴定。接种菌为上海地区优势黄瓜白粉菌生理种,经单孢分离和纯化。共鉴定出高抗材料17份,耐病材料(中抗,中感)82份,高感病材料21份。高抗材料包括中国10份,中国台北、日本各2份,美国、荷兰、菲律宾各1份。本试验为培育黄瓜抗病品种和跨种植区引种,提供了良好的技术支持。2.以华北类型黄瓜自交系S94(感白粉病,母本)和欧洲温室型黄瓜自交系S06(抗白粉病,父本)为亲本材料,配置F6群体,构建了一张黄瓜分子标记遗传图谱。该图谱包括7个连锁群,含有257个标记,包括206个SRAP (sequence-related amplified polymorphism)标记,22个SSR(simple sequence repeat)标记,25个SCAR(sequence charact- erized amplified region)标记,1个STS(sequence tagged site)标记和3个形态学标记,图谱总长度1005.9cM,标记平均间距是3.9cM。3.对黄瓜自交系S94和S06及其由它们构建的130个F2:3家系,分别利用温室人工喷雾接种法(2005秋、2006春)和叶盘法(2005秋)进行白粉病抗性鉴定;并使用该群体对应的分子标记连锁图进行数量性状座位(quantita -tive trait loci,QTL)分析。使用复合区间定位方法共检测到5个白粉病抗性QTLs(pm1.1,pm1.2,pm2.1,pm2.2和pm5.1),分布于连锁群1、2和5,单个QTL的贡献率介于3.4%～45%之间。其中pm1.1,pm2.1和pm5.1在温室人工喷雾接种法和叶盘接种法都能检测到。4.同时还利用黄瓜自交系S94和S06及其由它们构建的224个F6:7家系的重组自交系(recombinant inbred lines,RIL)群体,分别在2005年秋和2006年春温室喷雾接种,进行白粉病抗性遗传分析;并在对应分子标记遗传图谱上,使用复合区间定位方法检测白粉病抗性QTLs。结果显示,在两种环境里共检测到黄瓜白粉病抗性的4个QTLs(rpm1.1、rpm2.1、rpm4.1和rpm6.1),分别分布于连锁群1、2、4和6上,单个QTL解释贡献率介于5.2%～21.0%之间,rpm4.1的表型贡献率最高(20.5%- 2005年秋,21.0% -2006年春)。其中rpm1.1、rpm2.1和rpm4.1在两种环境中均被稳定重复检测到,rpm6.1只在2005年秋被检测到。两种环境下检测的QTLs解释表型变异总和分别是52.0%(2005秋)和42.0%(2006春)。把F6:7与F2:3群体定位的白粉病QTL相比较发现,rpm1.1和rpm2.1分别与pm1.2和pm2.1附近的标记相同,应该为相同的QTL。但不同世代群体间相同的QTL,存在表型变化贡献率和加性等位基因来源方向不一致的现象。生产实践中,杀菌剂也被大量用于植物病害防治。使用杀菌剂,一方面增加了病原小种的变异率,另一方面表现出环境不友好和食品污染等问题。为了饮食健康,研究人员积极开发寻找高效、环保的控病措施。已有报道,Vitamin B1能作为激发子,诱导拟南芥、水稻和烟草产生抗病性。本研究对Vitamin B1作为激发子提高黄瓜白粉病抗性的作用机理进行了初步研究。1.通过考察Vitamin B1处理黄瓜幼苗白粉病发病的病情指数,确定50mmol/L Vitamin B1为最佳处理浓度,此浓度处理比对照水处理后的病情指数减少38%。2.用50mmol/L-Vitamin B1处理黄瓜感白粉病自交系S52第1片真叶,4h后第1片真叶接种黄瓜白粉病菌,分时间段取心叶提取叶片RNA,利用实时荧光定量PCR检测病程相关标记基因[病程相关蛋白-1a(pathoge-nesis-related protein-1a,PR-1a)基因、过氧化物酶(acidic peroxi -dase,POX)基因、脯氨酸裂解酶(phenylalanine amonia-lyase,PAL基因]与对照水处理0h相比的表达变化。结果发现3个基因72h内都表现出表达先快速增加,后降低的变化;表达量最大变化,分别出现在12h、48h和12h;分别是水对照的3.1、4.9和2.5倍。结果初步表明Vitamin B1预处理黄瓜自交系S52幼苗,能引起系统获得抗性(systemic acquired resistance,SAR)反应,使S52获得对白粉病的系统抗性。更多还原

【Abstract】 Powdery mildew (PM), caused by Podosphaera xanthii (formerly known as Sphaerotheca fuliginea Schlech ex Fr.Poll.) or Golovinomyces cichoracearum (formerly known as Erysiphe cichoracearum DC ex M/erat.), limits the production of cucumber (Cucumis sativus L.) throughout the world. In China, Podosphaera xanthii has been identified as the primary cause of PM in both the greenhouse and field cucumber cultivation. The PM pathogens have a wide host range and are readily detached and borne by air currents. According to the investigation, the severe PM infection occurred before the flowering stage could reduce the output of the fresh cucumber fruits by 30% to 50%. Developing resistant cultivars would provide an economic and evironment-friendly control strategy. Although former studies have showed that the resistance or susceptibility to PM infection varies among the cucumber cultivars, up to now, there have been few evidences for the existence of the single gene-controlled high resistance to PM in the surveyed cucumber varieties. Thus, it would be a hard task to breed high resistance cucumber lines to PM by traditional breeding method. To research the principle of PM resistance and locate the resistance gene, which is very important for actualizing molecular-marker assisted selection breeding and cloning resistance gene in the future.1.By means of leaf-inoculation by two repetition test, the resistance of 120 germplasm of cucumber to cucumber powdery mildew were identified and evaluated with resistance index. The results indicated that among the tested 120 materials, high-resistant, mid-resistant and mid-susceptive, susceptive materials were 17, 82 and 21 strains, respectively. High-resistant materials come from ten of China cucumber materials, in addition, including each two from China Taiwan and Japan, each one contribute by America, Holland and Philippine. The important resistance disease informations about cultivating elicit cucumber variety were offered through the experiment.2.We constructed F6∶7 population from resistant line S06 as male parent cross with sensitive line S94(Northern China open-field type) as female parent. The linkage map used in this study using molecular marker technique constructed, includes 257 molecular markers [206sequence-related amplified polymorphisms (SRAP), 22 simple sequence repeats (SSR), 25sequence characterized amplification regions (SCAR), 1 sequence-tagged site (STS) and 3 morphologic traits ] with the total map length of 1005.9 cM and a mean marker interval of 3.9 cM, which is suitable for QTL analysis.3. PM is an economically important disease of cucumber caused by the obligate biotrophic pathogen, Podosphaera xanthii, in greenhouse and field. In this study, a resistant breeding line S06 and a susceptible cucumber inbred line S94 and their F2:3 populations were used to investigate PM resistance under seedling spray inoculation in two growing seasons and leaf disk infection in one season. QTL analysis was undertaken based on a constructed molecular linkage map of the corresponding F2 population using composite interval mapping. Five QTLs (pm1.1,pm1.2, pm2.1, pm2.2 and pm5.1) for PM resistance were identified and located on linkage groups (LG) 1, 2 and 5, explaining 3.4～45% of the phenotypic variation. Three consistent QTLs (pm1.1, pm2.1 and pm5.1) were detected under the three test conditions.4. S06 and S94 and their F6:7 populations were also used to investigate PM resistance under seedling spray inoculation in 2005/Autumn and 2006/Spring. QTL analysis was undertaken based on a constructed molecular linkage map of the corresponding F6 population using composite interval mapping. A total of four QTLs (rpm1.1, rpm2.1,rpm4.1 and rpm6.1) for PM resistance were identified and located on LG 1, 2, 4 and 6, explaining 5.2%～21.0% of the phenotypic variation. Three consistent QTLs (rpm1.1, rpm2.1 and rpm4.1) were also detected under the two test conditions.The rpm6.1 was only identified in 2005/Autumn. The total phenotypic variation explained by the QTLs was 52.0% and 42.0% in 2005/Autumn and 2006/Spring,respectively.According to the data from the two different generations collectively, only two QTLs were consistent on LG1 and LG2, respectively. The position of additive alleles and phenotypic variation of these QTLs were variance between F2:3 and F6:7.Disease control relies mainly on the extensive use of fungicides, with the risk of development of resistant pathogen strains. To obtain green food, the replacement of fungicides by effective and environmentally friendly methods is the major way forward. Vitamin B1 was also reported to have medicinal uses in Arabidopsis and rice. In the present experiment, we invest -igated the efficacy of Vitamin B1 which protected cucumber seedling against the cucumber powdery mildew fungus and the resistance disease mechanism for Vitamin B1 operation.1.Pre-treatment of the first leave of cucumber seedlings with Vitamin B1, four hours prior to inoculation with powdery mildew, reduced the disease index by over 38% at 50 mmol/L ,which was confirm prime treatment concentration.2.Using 50 mmol/L Vitamin B1 and powdery mildew(Podosphaera xanthii ) to dispose cucumber first true leaf and take hearts leaf RNA according to time level, pathogensis-related defense marker gene[pathogenesis-related protein-1a, (PR-1a) acidic peroxidase(POX), , phenylalanine ammol/Lonia -lyase (PAL) ]expression with contrast control(water treatment,0h) were checkout through real time’s fluorescence PCR. Results showed that in inocul -ated plants that measured increases in POX, PR-1a and PAL expression appeared highest at 12h,48h and12h post treatment,respectively (an approxi -mate 3.1-fold increase in PR-1a expression, 4.9-fold increase in POX expre -ssion and 2.5-fold increase in PAL expression). The results showed that pre -treatment of cucumber seedlings with Vitamin B1 can gain resistance to powdery mildew through systemic acquired resistance (SAR) track.更多还原