伯克氏菌属和葡萄座腔菌属植物病原菌DNA条形码及分子检测研究

发布时间：2018-02-24 16:22

本文关键词： 伯克氏菌葡萄座腔菌 DNA条形码检测鉴定　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：DNA条形码技术是一种物种快速鉴定的方法,受到广泛关注,能够弥补传统形态学鉴定的不足,推动生物多样性研究的发展。伯克氏菌属(Burkholderia)属于变形菌门(Proteobacteria)是一类重要的革兰氏阴性细菌,报道超过60个种,广泛存在于土壤、空气及水体等环境中,部分种是动物或者植物重要的病原菌,其中包括B. gladioli pv. alliicola等在内的至少3种被列入中国进境检疫性有害生物名录。葡萄座腔菌属(Botryosphaeria)及其相关的无性世代所在的属分类复杂,可以用于分类的形态学特征较少且不稳定,难以通过形态学对其进行准确鉴定。基于以上背景,本研究以伯克氏菌属和葡萄座腔菌属为对象,开展了DNA条形码筛选及分子检测相关研究,主要结果如下：(1)伯克氏菌属DNA条形码研究:以伯克氏菌属29个种87株菌为材料进行了DNA条形码研究。以16S rRNA、gyrB、rpoD和lepA为候选基因,通过遗传距离分析、系统发育树构建和’TaxonDNA鉴定效果等方法对这4个候选基因进行了评价,结果表明16S rRNA基因种内和种间变异较小,gyrB基因种内差异最大,rpoD基因种问差异最大。基于系统发育树和’[axonDNA法鉴定成功率结果表明,lepA基因的鉴定成功率最高,可以作为伯克氏菌属DNA条形码,但是对于洋葱伯克氏菌复合种的鉴定能力需要进一步进行验证。以伯克氏菌属lepA基因为研究对象,比较DNA条形码方法,遗传距离法,基于距离的分析方法(ABGD法),基于聚类的方法(单系法)和基于特征的方法(BLOG法)。结果表明,特征法的对于75株菌的鉴定成功率为100%,高于其他三种方法,对于洋葱伯克氏菌复合种也能够有效的进行鉴定。(2)洋葱腐烂伯克氏菌的LAMP检测;针对洋葱腐烂病菌(.B. gladioli pv. alliicola) ITS序列设计了4条LAMP引物,通过优化反应温度,建立了实时LAMP及可视化LAMP检测方法。该方法特异性好,仅对Bga菌株检测呈阳性,对于与Bga同属不同种或同种不同致病型菌株,检测结果均为阴性。检测灵敏度达到1fg/μL的基因组DNA,比普通PCR灵敏度高100倍,能够用于洋葱腐烂病菌的快速检测。(3)葡萄座腔菌属DNA条形码研究：以ITS、LSU、TUB和EF-la为候选DNA条形码,利用遗传距离分析、系统发育树构建、TaxonDNA法和特征法对4个候选基因进行综合评价,结果表明,LSU基因种内和种间变异较小,EF-1α基因种内和种间差异最大。基于‘TaxonDNA法和特征值法鉴定结果表明,EF-1α基因的鉴定成功率最高,可以作为葡萄座腔菌属及其相关菌株的DNA条形码。(4)引起苹果腐烂病菌的三种葡萄座腔菌的实时荧光PCR检测：通过设计三组引物和探针,建立了快速检测三种苹果腐烂病菌B. dothidea, B. obtusa和B. stevensii的实时荧光PCR检测方法,检测灵敏度分别为10-3 ng/μL、10-3 ng/μL、10-4ng/μL。基于单一靶标的实时荧光,进一步构建了多重实时荧光PCR检测方法,检测灵敏度为10-3 ng/μL,能够实现同时对三种病原真菌的检测。
[Abstract]:DNA barcode technology is a rapid identification method of species, which has attracted wide attention and can make up for the shortcomings of traditional morphological identification. Burkholderiabelonging to Proteobacteria, an important class of Gram-negative bacteria, has been reported for more than 60 species in soil, air and water environments. Some species are important pathogens of animals or plants, and at least three of them, including B. gladioli PV. Alliicola and others, are included in the list of quarantine pests in China. The taxonomy of the genus Botryosphaeria and its related asexual generations is complex. The morphological characteristics that can be used for classification are less and unstable, so it is difficult to identify them accurately by morphology. Studies on DNA barcode screening and molecular detection were carried out. The main results were as follows: (1) DNA barcode study was carried out. DNA barcode was studied with 87 strains of 29 species of Burke's genus. 16s rRNAs gyrBrpoD and lepA were used as candidate genes. The four candidate genes were evaluated by genetic distance analysis, phylogenetic tree construction and Taxon DNA identification. The results showed that the variation of 16s rRNA gene within species and between species was the greatest. Based on phylogenetic tree and'[axonDNA] method, the results showed that the success rate of identification of rRNA A gene was the highest. It can be used as DNA bar code, but the identification ability of Burke's bacteria complex species needs further verification. Taking lepA gene of Burke's bacillus as the research object, the method of DNA bar code and genetic distance method are compared. Distance based analysis methods include ABGD method, clustering method (single line method) and feature based method (blog). The results show that the success rate of characteristic method for 75 strains of bacteria is 100, which is higher than that of the other three methods. The complex species of Burkella cepacia could also be effectively identified by LAMP, and four LAMP primers were designed for the ITS sequence of B. gladioli pv.alliicola, and the reaction temperature was optimized by optimizing the reaction temperature. A real-time LAMP and visual LAMP detection method was established. The method was specific and positive only for Bga strains, and was different from the same species or the same pathotypes as Bga. The genomic DNA with a sensitivity of 1 fg / 渭 L was 100 times higher than that of the common PCR, and could be used for the rapid detection of the onion rot bacteria. DNA bar code was used for the study of the genus DNA: ITSLSUTUB and EF-la were used as candidate DNA bar codes. Using genetic distance analysis, phylogenetic tree construction Taxon DNA method and characteristic method were used to evaluate the four candidate genes. The results showed that the variation of LSU gene within species and between species was the greatest, and the result of Taxon DNA and eigenvalue method showed that the success rate of identification of EF-1 伪 gene was the highest. It can be used as the DNA bar code of the genus Vitis and its related strains. It can be used to detect three kinds of rotting bacteria of apple by real-time fluorescence PCR: by designing three sets of primers and probes, A real-time fluorescence PCR method for rapid detection of three kinds of apple rot bacteria B.dothidea, B. obtusa and B. stevensii was established. The detection sensitivity was 10-3 ng / 渭 L ~ (10 ~ (-3)) ng / 渭 L ~ (10 ~ (-4)) ng / 渭 L. based on a single target, a multiplex real-time fluorescence PCR detection method was further constructed. The detection sensitivity was 10-3 ng / 渭 L, which could be used to detect three pathogenic fungi simultaneously.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.4

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