【作者】 刘芳；

【导师】 刘红霞；

【作者基本信息】 北京林业大学 ， 森林保护， 2010， 硕士

【摘要】 非洲菊(Gerbera jamesonii Bolus)又名扶郎花,菊科(Asteraceae)大丁草属(Gerbera)多年生草本花卉,为世界五大鲜切花之一。非洲菊叶斑病、白粉病和根腐病的发生蔓延,给北京及周边地区非洲菊花卉产业带来很大影响。因此明确病害的病原菌,寻找非洲菊中与抗病相关的基因,对于防治病害具有重要意义。本研究利用科赫氏法则对非洲菊主要病害的病原菌进行回接鉴定,并通过同源序列克隆法得到非洲菊的抗病基因同源序列(Resistance Gene Analogs, RGAs),然后分析RGAs与抗病基因的关系,进而确定候选抗病基因。通过上述研究可得到以下结果：1.利用离体叶片接种法对非洲菊叶斑病病原菌进行回接验证,采用形态学结合分子生物学的方法鉴定该病原为：丝孢纲丝孢目暗色孢科链格孢属链格孢(Alternaria alternate (Fr.) Keissl)和细极链格孢(Alternaria tenuissima (Fr.) Wiltshire),将ITS序列提交GenBank得到登录号：GQ169728和GQ169727。2.利用根部刺伤和无伤接种对非洲菊根腐病病原菌进行回接验证,采用形态学结合分子生物学的方法鉴定该病原为：丝孢纲瘤座孢目瘤座孢科镰孢属木贼镰刀菌(Fusarium equiseti(Corda) Sacc),将ITS序列提交GenBank得到登录号：HM008677。3.利用自然接种法和人工接种法对非洲菊白粉病病原菌进行验证,采用形态学结合分子生物学的方法鉴定该病原为：核菌纲白粉菌目白粉菌科的Podosphaera fusca(Fr.) U. Braun & Shishkoff(异名：P. xanthii、P. balsaminae、Sphaerotheca fusca和Sfuliginea),将ITS序列提交GenBank得到登录号：HM002584。此外,对P. fusca分生孢子的萌发生物学特性进行了研究,发现孢子萌发的适宜温度范围是22℃-25℃,尤其以25℃最为合适,pH为6时萌发率显著高于其它pH条件,水滴可能是非洲菊白粉病菌萌发的必要条件。采用沈阳农业大学纪明山教授提供的蜡蚧轮枝菌制剂对非洲菊白粉病进行盆栽防治试验发现,制剂浓度为2.6g／L～3.3g/L时能有效防治非洲菊白粉病的发生。4.以“红色恋人”和“水粉”的基因组DNA为模板,根据R基因的保守区域设计9对引物进行PCR扩增,筛选出3对合适的非洲菊基因组扩增引物,获得了4条长度分别为250bp、2000bp、700bp和2500bp的扩增条带,回收条带进行连接转化,蓝白斑筛选,随机挑选菌落进行PCR鉴定,共得到30个阳性克隆。5.将这30个阳性克隆进行测序和同源性搜索,结果表明有15个阳性克隆片段与R基因具有较高的同源性。进一步对序列进行分析,发现有9个阳性克隆片度具有完整开放阅读框。对这9条RGAs的推导氨基酸序列进行聚类分析,推测这9条非洲菊RGAs可分为TIR-NBS-LRR和non-TIR-NBS-LRR两个亚类,证实了以往报道的双子叶植物中存在两类NBS-LRR基因的结果。将不具备完整开放阅读框的RGALR1的3’和5’端编码的氨基酸进行分析时发现共具有8个LRR重复。更多还原

【Abstract】 Gerbera jamesonii Bolus is one of the most famous cut flowers in the world, which belongs to family Asteraceae. However, gerbera leaf spot, powdery mildew and root rot speading in Beijing and surrouding areas cause great economics loss to the growers. Clarify disease pathogens and find disease resistance genes mean a lot to prevent and cure gerbera disease. This study mainly identified pathogens of three gerbera diseases using Koch’postulates, cloned Resistance Gene Analogs (RGAs) of gerbera, and analized the relationship between RGAs and resistance genes. The main research conclusions were shown as follows:1. The pathogens of gerbera leaf spot disease, identified on the basis of pathogenic test as well as morphological character and molecular identification, were taxonomically described as Alternaria alternata (Fr.) Keissl and Alternaria tenuissima (Fr.) Wiltshire, GenBank accession numbers are GQ169728 and GQ169727.2. The pathogen of gerbera root rot disease, identified on the basis of pathogenic test as well as morphological character and molecular identification, was taxonomically described as Fusarium equiseti (Corda) Sacc, GenBank accession numbers is HM008677.3. The pathogen of gerbera powdery mildew disease, identified on the basis of pathogenic test as well as morphological character and molecular identification, was taxonomically described as Podosphaera fusca (Fr.) U. Braun & Shishkoff (also called P. xanthii、P. balsaminae、Sphaerotheca fusca and S. fuliginea), GenBank accession numbers is HM002584. Trials determined the teliospores germinating biology of P. fusca to provide basis for control teliospores germinate in order to avoid the disease, by the way of adjusting ecology environment. The optimum teliospores germinating temperature range was 22℃to 25℃, especially 25℃. Germinating rate was extraordinarily high when pH was 6, and water may be necessary for the teliospores germination. The effectiveness of Verticillium lecanii preparation as curatively applications in the control of gerbera powdery mildew was tested, the result showed that conidiophores concentration 2.6g/L-3.3g/L of V. lecanii could control gerbera powdery mildew disease more effectively.4. Specific fragments of 250bp、2000bp、700bp and 2500bp were cloned from genomic DNA of Hongselianren and Shuifen by PCR with three-pair primers out of nine designed according to the conserved domains of reported Resistance Genes. Before transformed into E.coli DH5a these specific fragments were recyled and inserted into pEASY-T3 vector. Thirty positive recombinants were obtained and confirmed through colony PCR identification.5. Fifteen out of these 30 sequenced clones had high homology contrast with reported R gene through blasting in GenBank, among which 9 RGAs had complete open reading frames (ORFs). Further cluster analysis showed that these 9 RGAs in gerbera may belong to TIR-NBS-LRR and non-TIR-NBS-LRR, which confirmed the previous study that two types NBS-LRR genes exist in dicotyledon. Eight LRR repeat units can be found in 3’and 5’ends of derived amino acid of LR1.更多还原